Mojica – Taipalus 0

The Effect of Hydrogen Peroxide and Duration of Soaking on the Growth of Escherichia

coli on Cork

Knicko Mojica and Dylan Taipalus

Macomb Mathematics Science Technology Center

Biology 9B

Mr. Acre/Mr. Estapa/Mrs. Gravel

19 May 2016
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Table of Contents

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

Problem Statement ...........................................................................................................................3

Experimental Design ........................................................................................................................4

Data and Observations ...................................................................................................................10

Data Analysis and Interpretation ...................................................................................................14

Conclusion .....................................................................................................................................22

Works Cited ...................................................................................................................................26

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Introduction

In the United States alone, almost 50% of students have played a musical instrument at

some point in their education (Cohen). Instruments including the saxophone and the clarinet

require expensive wooden reeds that need tedious attention and care to help preserve their quality

reduce the spread of bacteria on the reed. The aim of the following experiment was to use a solution

that does not hinder the playability of the reed while reducing the growth of bacteria on the reed.

Wood cork was used in place of wooden reeds to reduce costs while still producing the same results

if the experiment were to be redone using wooden instrument reeds.

The goal of the experiment was to produce the greatest zone of inhibition of Escherichia

coli after exposing a thin slice of wood cork to varying concentrations of hydrogen peroxide (H2O2)

for varying periods of time in minutes. Six thin slices of cork, cut at 3 mm thin, were placed into

dishes of varying hydrogen peroxide concentrations for different times. Two of the cork slices

were placed into one dish to conduct more standard trials as compared to the other trials. The set

Petri dishes are placed into an incubator at 37 °C for 24 hours. The zone of inhibitions around the

cork were measured the next day in millimeters. This process was repeated nine times.

Little to no prior research has been done on the sanitization of cork reeds, however, prior

research noting safe concentrations of hydrogen peroxide and its non-toxic qualities have been

conducted. Very few articles measured the reduction of E. coli by measuring zone of inhibition.

“Escherichia coli K12 was soaked in varying concentrations of hydrogen peroxide for fifteen

minutes. Cells were more susceptible to lower concentrations of hydrogen peroxide (less than

three mm) as compared to intermediate (five to twenty mm) concentrations. When the solution

was greater than twenty mm, cell survival was inversely proportional to concentration.” (Linely

et al.). The article described the effects of hydrogen peroxide on E. coli after soaking for 15
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minutes, which was the standard soaking time used in this experiment. The research conducted in

the article also measured E. coli reduction by measuring zone of inhibition, which is the method

used in this experiment. Other articles and research discussed the uses for hydrogen peroxide but

few discussed exposure to E. coli.

As the concentration of hydrogen peroxide increases, the zone of inhibition, measured in

millimeters around the soaked corks increases. As concentrations decrease, the zone of inhibition

around the cork decreases in size. The concentrations should not extend beyond 3% and 12%

because concentrations below 3% will yield little to no results while anything higher than 12% is

not commercially available and is not safe for everyday use (Rutala and Weber).

As the duration of time that the cork soaking increases, the zone of inhibition around the

cork increases. As the duration time decreases, the zone of inhibition will decrease. The

concentrations should not exceed 22 minutes and 8 minutes because exceeding 22 minutes will

overwhelm or oversoak the cork. If the cork is soaked for less than 8 minutes, the hydrogen

peroxide will not have soaked the cork for long enough, producing little to no results (Rutala and

Weber).

Since the experiment aimed to produce the largest decrease in bacterial growth, it was

decided to measure the average zone of inhibition around the cork in order to see sufficient results.

While there are alternative methods to measuring bacterial growth, measuring the zone of

inhibition was the best option to record data for this experiment.
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Problem Statement

Problem:

To determine the effect of hydrogen peroxide and duration soaking on the growth of

Escherichia coli on cork.

Hypothesis:

If E. coli is exposed to 12% hydrogen peroxide for 22 minutes, then there will be a larger

one of inhibition around the cork as compared to a cork soaked for 8 minutes in 3% hydrogen

peroxide.

Data Measured:

Since hydrogen peroxide is known as an alternative sanitizing agent, it was chosen as one

of the independent variables to be tested. The other variable that will be tested is the amount of

exposure time the E. coli exposed cork will have. The first standard variable is a 1:1 ratio of a

hydrogen peroxide and distilled water solution, based on a 5 mL total solution. The second

standard variable is the amount of exposure time which is fifteen minutes. Based on prior

studies, the ratio of the hydrogen peroxide solution was chosen because it would overpower the

other independent variable. The exposure time was chosen to be fifteen minutes based on a study

facilitated by the Center for Disease Control which stated that the average time for E. coli to be

killed by hydrogen peroxide was fifteen minutes, "(e.g., E. coli, Streptococcus species, and

Pseudomonas species) required only 15 minutes’ exposure" (Rutala and Weber). The high is a

twenty-minute exposure and a 2:1 ratio of hydrogen peroxide to water because it is high enough

to differentiate from the standard while not going too high. The low is a ten-minute exposure and

a 1:2 ratio of hydrogen peroxide to water because these are the minimums before the

independent variables would not have an effect.

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Experimental Design

Materials:
(100) 10 mm x 7 mm Bottle Corks (1) 3.79 Liter Distilled Water
(1) 946 mL 3% Hydrogen Peroxide (H202) (1) 473 mL 12% Hydrogen Peroxide (H202)
(40) Petri Dishes 23 g of Agar
(1) 1 mL Dropper Escherichia coli
TI-Nspire Calculator (1) Ruler (millimeter)
(1) Notebook (1) Pen/Pencil
(1) 250 mL Test Tube (1) Transfer Loop
(1) Pipette (1) Stirring Magnet
(1) 10 mL Graduated Cylinder (1) Sharpie Marker
(1) Clock/Timer (1) Incubator set at 37 °C
(6) 40 mL Glass Dishes (1) 18.5 m2 Plastic Wrap
(1) Large Pot of Boiling Water (1) Pair of Tongs
(40) 15 cm x 15 cm Paper Towel (1) Large Box Cover

Procedure:

Disclaimer: This procedure is set-up as if the trials were to be done in one day. It is
recommended that five trials be done each day, each one pertaining to each DOE
group [(-,-), (-,+), (s,s), (+,-), (+,+)]. The same steps still apply.
Agar Prep:

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

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

3. Add a stirring magnet to the 1 liter of water. Place the magnet on setting #4

4. Slowly add 23 grams of agar powder to the flask, being careful not to get the powder on
the sides

Note: A full 1-liter batch will fill 50 Petri dishes

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

6. Remove from heat and allow to cool in order to be able to pour it

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Agar Pouring:

Note: When pouring the agar into a Petri dish, do not open the cover too wide or expose the
inside of the Petri dish to the open air. Also, never open the lid and place it onto the
tabletop.

7. Label the dish with the date, names, and E. coli and open the lid of the Petri dish just
enough to pour a thin layer (1-3 mm thick) of agar

8. Close the lid and allow the agar to cool until it appears to resemble a jello-like substance

9. When cool, add the E. coli (Refer to the Transferring E. coli to the Petri Dish section)

Transferring E. coli to the Petri Dish:

10. With one test tube, fill it with 1 mL of distilled or bottled water using a pipette

11. Place the test tube aside, keeping it upright

12. While holding the E. coli sample, take the inoculating loop and drop it into the E. coli
sample

Note: Before using the transfer loop, sanitize it above an open flame and allow to cool for 10
seconds before each use

13. Take the transfer loop, and drop it into the test tube filled with 1 mL of distilled water
and move it around in the water to mix the E. coli

14. Remove the transfer loop and carefully pour the E. coli solution into the agar prepared
Petri dish

15. Swish the Petri dish around for around 30 seconds, making sure that the E. coli is evenly
spread around the agar

Note: Be sure to keep the Petri dish lid closed at all times to reduce the risk of contaminating
the Petri dish

16. Place dishes into incubator set at 37 °C for 24 hours

17. Repeat steps 7 through 16 until 30 agar dishes are prepared

Cork Prep:

18. Open bag of corks

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19. Take out 1 of the 40 mL glass dishes and set it to the side, making sure not talk over it or
get any other contaminants inside, as the dish must remain as sterile as possible

20. Using the razor, cut a 3 mm slice of cork

Note: Three slices of cork may be used per each whole cork piece

21. Set the slices of cork into the glass dish

22. Dispose of the rest of the cork

23. Repeat steps 17 through 21 until there are 40, 3 mm cork slices in the glass dish.

24. Set aside the cut corks, cover the glass dish with plastic wrap to avoid contamination

Solution Prep:

Note: Before pouring solutions into glass dishes, place the dishes into a pot of boiling water in
order to sanitize them, leave them in the water for 1 minute. Use tongs to remove the
dishes and handle them carefully

25. Have 5 sterile glass dishes out for use. On 5 pieces of paper towel, label each one with
the corresponding low and high. [ex. (+,-) or (12% H O , 8 minutes)]
2 2

26. Remove the cap off of the bottle of 3% Hydrogen Peroxide (H 0 ) and pour 5 mL of H 0
2 2 2 2

into a graduated cylinder

27. Pour the H 0 from the graduated cylinder into the (-,-) glass dish
2 2

28. Repeat step 26 but instead pour the solution into the (-,+) glass dish

29. Remove the cap off the 12% and pour 2.5 mL into the graduated cylinder

30. Pour the 12% H O into the (s,s) glass dish

2 2

31. Using the same graduated cylinder, open the distilled water and pour 2.5 mL into it

32. Pour the distilled water into the (s,s) glass dish and swish the solution to mix it

33. With the 12% H O , pour 5 mL into the graduated cylinder

2 2

34. Pour the H O into the (+,+) glass dish

2 2

35. Repeat step 33 but this time, pour the H O into the (+,-) glass dish
2 2

36. Cover solutions with the box lid to reduce exposure to light
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Exposing the Cork to the Hydrogen Peroxide Solution and E. coli:

Note: When exposing the corks to the hydrogen peroxide and E. coli, keeping the corks
uncontaminated is of utmost importance. Keep the area as clean and as organized as
possible.

37. Prior to placing the cork in the solutions, use the random integer function on the TI-
Nspire (using the number 1 through 5) to determine the order in which you will place the
cork in the designated solution. [(-,-) = 1, (-,+) = 2, (s,s) = 3, (+,-) = 4, (+,+) = 5]

Note: Be sure to understand which solution and submersion time is being used; (-,-) = (3%, 8
min), (-,+) = (3%, 22 min), (s,s) = (6%, 15 min), (+,-) = (12%, 8 min), (+,+) = (12%, 22
min).

38. Grab one cork from the cup and place it in the solution indicated by the calculator

39. Once the cork is placed into the solution, immediately start the timer for the designated
time

40. Once the timer goes off, grab the cork from the solution and place it in Petri dish and put
the lid on to the dish

Note: Do not touch the agar or inside of the dish with anything except for the soaked cork

41. Repeat steps 36 through 39 until all solutions have been used and the 40 dishes of agar
have been filled

Note: When transferring the cork to the solution, keep the box lid covering the solutions as
much as possible.

Incubating the Cork:

42. Stack all the Petri dishes on top of each other

43. Place all of the dishes into an incubator set at 37 °C, keeping the side of the dish that is
labeled, towards the bottom so that the cork stays in its position

44. Let the dishes sit in the incubator for 24 hours

45. After 24 hours remove the Petri dishes from the incubator and lay them all down with the
labeled face showing up

Note: After incubation, the cork will be stuck to the agar, allowing for the dishes to be flipped
over.
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Recording Data:

46. Set up 5 columns in a notebook page

47. Label the columns “(-,-)” “(-,+)” “(s,s) “(+,-)” and “(+,-)”

48. Pull the Petri dishes out of the incubator

49. Set the dish on the work space with the lid facing up

50. Measure the diameter of the zone of inhibition for each disk. Keeping the lid of the plate
in place, use a ruler to measure the diameter of the cork plus the surrounding E. coli-free
area in millimeters (mm). Include the diameter in the notebook. Be sure to record the
right date in each column

51. Repeat steps 46 through 50 until all of the corks’ zone of inhibitions have been recorded

Clean up:

52. Any materials used in the experiment that came in contact with the E. coli must be
thrown out, excluding the test tubes which can be washed

53. Any other materials that were used for solutions or agar prep can either be washed or
disposed of

54. If any materials were not used in the experiment then they can be placed in a cabinet to
be used for later experiments

55. Wipe down the workspace used with either disinfectant wipes or paper towels and
disinfectant spray
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Diagram:

Figure 1. Experiment Materials

Figure 1 shows the necessary materials to complete the experiment, including the

hydrogen peroxide solutions and cork which will be tested

on E. coli.
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Data and Observations

Data:
Table 1
Design of Experiment Values
H2O2 (%) Duration (minutes)
- Standard + - Standard +
3 6 12 8:00 15:00 22:00

Table 1 above shows the two variables used in the experiment (hydrogen peroxide percent

and duration soaking in minutes). The standards are set at 6% hydrogen peroxide and 15 minutes

soaking because it is proven that Escherichia coli is killed by 6% hydrogen peroxide in 15 minutes

or less. The hydrogen peroxide solutions and duration soaking were chosen so that the E. coli was

not completely killed and still produced results.

Table 2
Data Collected
Zone of Inhibition (mm)
DOE (Solution %, Duration min.)
(+,+) (+,-) (-,+) (-,-)
1 57 38 27 17
2 56 41 26 16
3 56 40 27 17
4 59 36 29 17
5 57 39 27 16
6 56 38 29 13
7 56 36 25 14
8 57 35 27 15
9 58 39 26 14
Average 57 38 27 15

Table 2 represents the DOE results of each of the nine trials, as well as their averages, in

their respective order.

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Table 3
Standard Results
18 Standards (mm)
23 21 22 25 26 23 23 20 20
21 21 22 21 26 23 23 20 20

Table 3 shows the results of the 18 total standards. The range of standards is 6 mm. In

relation to the other sets of data (ex. (-,-) ,(+,+), etc.) the range of standards of 6 mm as compared

to the average range between the other DOE results of 4.25 mm is very close.

Observations:

Table 4
Observations
Date Observation
3/08/2016 First trial runs smoothly, no errors found
3/10/2016 Cork in standard Petri dish moved to the side; zone of inhibition is still present
3/15/2016 Mold growth is present in all the dishes; zones of inhibition is still present
Corks stuck to the top of Petri dishes (smaller Petri dish used), not allowing the
3/17/2016
H2O2 to spread; trials redone
3/18/2016 (-,+) and (+,-) are soaked for incorrect times; trial redone next day
3/22/2016 Cork is cut with a razor instead of a scalpel; cork is cut more precise
3/24/2016 Multiple DOE trials are setup and run to catch up on behind trials
3/29/2016 Cork in (-,+) has a very small zone of inhibition; trial redone

Table 4 contains notes on observations and anything that was not expected during the trails.

The mold growth and cork moving were unexpected but did not affect the trails. Corks were also

cut slightly smaller to prevent the corks from sticking to the top of the Petri dishes.
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Cork moved away

from center of Petri
dish

Yellow/Orange mold
growth around cork

Figure 2. Mold Growth and Moved Cork

Figure 2 shows the growth of mold which is very visible in the (s,s) and (+,+) Petri dishes.

In the (s,s) Petri dish, the cork has moved to the top, but the zone of inhibition is still present.
 Mojica – Taipalus 13

The zone of inhibition is smaller

than usual because the cork was
stuck to the top of the Petri dish

Figure 3. Cork Stuck to Top of Petri Dish

Figure 3 shows the resulting smaller zone of inhibition of the (-,+) Petri dish after the cork

was stuck to the top of the dish instead of completely resting on the Escherichia coli. For this trial,

smaller Petri dishes were used as compared to the other trials, making the cork more susceptible

to sticking to the top of the dish lid. The trial was redone.
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Data Analysis and Interpretation

A Design of Experiment, or DOE with two factors was used to analyze the data in the

experiment. The DOE was used to test how two variables and their interactions would affect a

response variable. In this experiment, cork was soaked in varying concentrations of hydrogen

peroxide (H2O2) for different times in minutes to see its effect on the zone of inhibition in a Petri

dish with Escherichia coli.

After placing the soaked cork in a Petri dish in an incubator set at 37 °C for 24 hours, it

was removed and placed onto a table where the zone of inhibition around the cork was measured

in millimeters using a ruler. Six trials were done each day, every other day over an 18-day period.

Four Petri dishes contained corks soaked in different concentrations of hydrogen peroxide for

different times. Two Petri dishes were standards. More standard trials were conducted to help

improve the accuracy of the experiment. Results were recorded onto a sheet of paper and pictures

were taken to document the results. There was little to no variance between each trial run,

eliminating chances of noise variables.

Table 4
Table of Factors
H2O2 (%) Duration (minutes)
(-) (S) (+) (-) (S) (+)
3 6 12 8:00 15:00 22:00

Table 4 above shows the two variables used in the experiment (hydrogen peroxide percent

and duration soaking in minutes). The standards are set at 6% hydrogen peroxide and 15 minutes

soaking because it is proven that Escherichia coli is killed by 6% hydrogen peroxide in 15 minutes

or less. The hydrogen peroxide solutions and duration soaking were chosen so that the E. coli was

not completely killed and still produced results.

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Table 5
Data Averages
Runs
1st 2nd 3rd 4th 5th 6th 7th 8th 9th
H2O2 Duration Avg.
DOE DOE DOE DOE DOE DOE DOE DOE DOE
(%) (min.)
+ + 57 56 56 59 57 56 56 57 58 57

- - 17 16 17 17 16 13 14 15 14 15

+ - 38 41 40 36 39 38 36 35 39 38

- + 27 26 27 29 27 29 25 27 26 27

Grand Average 34.25

Table 5 above shows the averages (in mm) from all nine trials. The (+,+), or 12 % hydrogen

peroxide and 22 minute soaking time produced the highest average zone of inhibition of 57 mm,

as indicated by the bolded text. This is a large difference between the other three DOE set-ups.

The table above also shows the grand average which was 34.25 mm.

18 Standards
28

24 26 26
25
Zone of Inhibition (mm)

20 23 22
23 23
22
23 23
21 21 21 21
20 20 20 20
16

12

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9
Trials
Figure 4. Scatter Plot of Standard
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Figure 4 shows the results from all 18 standards taking place over the 9 days of trials.

Given the nine-day setup for the trials, no significant pattern can be seen, although, some of the

data from days 2, 3 and 7 were higher than the rest days. This was most likely due to the

temperature on the nights of those trials: the incubator was known for being effected by the

temperature in the room at night so that could have effected trials days 2, 3 and 7. The range of

standards is 6 mm, which means that the Range of Standards (ROS) multiplied by 2 is 12 mm.

Knowing this gives the basis for the test of significance.

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Table 6
Effect of Hydrogen Peroxide Effect of H2O2

Zone of Inhibition (mm)

H2O2 (%) 60
50 47.5
(-) 3% (+) 12%
40
15 57
30
27 38 21
20
Avg. = 21 Avg. = 47.5
10
Effect Value: 15.5 mm
0
-1 1
H2O2

Figure 5. Effect of Hydrogen Peroxide

Table 3 shows the average between both high and low trials in regards to the hydrogen

peroxide concentrations in millimeters (mm). The 12% hydrogen peroxide had a zone of

inhibition that was 26.5 mm larger than the zone of inhibition present in the 3% hydrogen

peroxide. The effect value represents the difference in the size of the zone of inhibition in

millimeters between the high, 12% value and low, 3% value. The effect value was calculated

by subtracting the (+) average minus the (-) average (47.5 – 21 = 26.5). The effect value was

statistically significant as well as practically significant because it passes the test of significance

due to how it’s larger than the ROS x 2 meaning this data was not received by chance alone. It

is practically significant because in the real world, no matter the solution of hydrogen peroxide

used, it will kill Escherichia coli. The positive slope of the segment in Figure 2 hints that as

the concentration of hydrogen peroxide increases, so will the zone of inhibition.

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Table 7
Effect of Duration Soaking Effect of Duration Soaking
60

Zone of Inhibition (mm)

Duration (min.)
50
(-) 8:00 (+) 22:00 42
40
15 57
38 27 26.5 30

Avg. = 26.5 Avg. = 42 20

Effect Value: 15.5 mm 10

0
-1 1
Duration

Figure 6. Effect of Duration Soaking

Table 7 represents the average between both high and low trials in regards to the duration

of time in minutes that the corks were soaked in millimeters (mm). The 22-minute soaking led to

a 15.5 mm larger zone of inhibition as compared to the 8-minute soaking. To calculate the effect,

the (+) average is subtracted from the (-) average (42 – 26.5 = 15.5). The effect value (15.5)

represents the difference in size of the zone of inhibition in millimeters between the high and low

value. The effect value is statistically significant because it passes the test of significance. The

positive slope of the segment in Figure 3 shows that as the time the cork is soaked increases; the

zone of inhibition will also increase. It is also practically significant because no matter how long

the cork is soaked; it will still kill Escherichia coli.

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Table 8
Effect of Hydrogen Peroxide and Effect of H2O2 & Duration
Duration Soaking Soaking

Zone of Inhibition (mm)

Duration (min) 60 57
Interaction of H2O2
(-) 8 (+) 22 50
and Duration
min. min. 38 40
30 27
Solid (+)
38 57
H2O2 (%)

Segment 12% 15
20
10
Dotted (-)
15 27 0
Segment 3%
-1 1
Interaction Effect 3.5 mm Duration

Figure 6. Interaction Effect of Hydrogen Peroxide

and Duration Soaking

Table 8 shows the average of the trials and their relationship to Figure 4 in millimeters

(mm). The solid line in Figure 4 represents the cork being soaked for 22 minutes while the dashed

line represents the cork being soaked for 8 minutes. 3.5 mm is interaction effect, or the interaction

between the two variables (Duration Soaking and H2O2). Since the slopes do not intersect in Figure

4, there is little to no interaction between the two variables. The interaction effect is not statistically

significant because the range of standards of 6 mm multiplied by 2 is greater than the interaction

effect of 3.5 mm (6 x 2 > 3.5). The positive slope of the segments in Figure 4, however, show that

as the duration that the cork is soaked increases or the concentration of hydrogen peroxide

increases, the zone of inhibitions will also increase.

If this interaction effect is compared to the overall effect of the H2O2 then it can be seen

that when it is held high, the average inhibition zone would be 47.5 mm. But, as seen above, when

duration is held low with H2O2 being held high, it drops nearly 10 mm. And when duration is held

high with the high H2O2 then the inhibition zone increases almost 10 mm. This goes the same when
 Mojica – Taipalus 20

the H2O2 is held low. This says that the high and low levels of H2O2 received their results regardless

of whether duration was held high or low

Dot Plot of Effects Legend:

HD: Interaction Effect of
H2O2 and duration
soaking
D: Effect of duration
D H soaking
HD 15.5 26.5 H: Effect of H2O2
3.5

-30 -24 -18 -12 -6 0 6 12 18 24 30

Figure 7. Dot Plot of Effects

The figure above shows the effect of H2O2 amount, duration of soaking, and the

interaction effect. The effects from both the H2O2 and soaking duration were both deemed

significant due to how they were both above the range of standards (ROS) multiplied by two,

which was 12 mm. More specifically, the H2O2 effect was 26.5 mm, 14.5 mm above the ROS x 2.

The effect of duration soaking was 15.5 mm, which was 3.5 mm above the ROS x 2. And finally,

the interaction effect was only 3.5 mm, which was 8.5 mm under 12 mm. Not only does this say

that the H2O2 has more of an effect on the killing of Escherichia coli than soaking duration does,

but it also says that the data from the trials could not have been from chance alone.

Parsimonious Equation
26.5 15.5
Y = 34.25+(s) +(d) + “noise”
2 2

Figure 8. Parsimonious Equation

The equation, above, allows for a prediction to be made, like the last equation, except it

leaves out any insignificant factors. In this instance, the interaction effect was removed due to how

it was below the 12 mm ROS x 2.

 Mojica – Taipalus 21

Prediction: (9%, 18.5min.)

𝟐𝟔.𝟓 𝟏𝟓.𝟓
Y = 34.25+(.5) +(.5) + “noise”
𝟐 𝟐

≈ 44.75 mm

Figure 9. Prediction Using Parsimonious Equation

The figure, above, would represent someone doing this same experiment but with using

9% H2O2 and a soaking duration of 18.5 minutes, which are both halfway in between the standard

and the high in this experiment.

Interpretation:

The effect of hydrogen peroxide and the effect of the duration of cork soaking both passed

the test of significance, deeming them statistically significant. Their interaction effect, however,

had little to no interaction and did not the pass the test of significance, deeming it statistically

insignificant. Even if their interaction is insignificant, each independent variable and the

interaction effect are practical in real world applications. No matter the concentration of hydrogen

peroxide or amount of time soaking, hydrogen peroxide will still kill Escherichia coli. This

experiment also helps to show that as the concentration of hydrogen peroxide and time soaking

increases, the larger the zone of inhibition will be as seen in the (+,+), or 12% hydrogen peroxide

and 22 minute soaking time trial.

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Conclusion

In the end, the effects of concentration of H2O2 and duration of soaking in the H2O2 effected

the Escherichia coli (E. coli) as hypothesized. It was hypothesized that the E. coli would produce

the highest inhibition zone when the cork was exposed to 12% H2O2 (+) and a 22-minute soaking

duration (+). Throughout the experiment, the sizes of the inhibition zone were recorded. By

looking at this data, it was seen that the groups that received the highest inhibition zone were the

trials that were exposed to the (+,+). Due to how much higher these inhibition zones were, the

hypothesis was fully accepted.

The goal of this experiment was to test the effect of H2O2 and duration of soaking on H2O2

for purposes pertaining to instrument reeds. Instead of a wooden instrument reed, wooden cork

was used because they were cheaper than reeds and would produce the same effects if reeds were

used instead. The cork would be soaking in varying H2O2 solutions (3%, 6%, 12%) and for varying

times (8 min, 15 min, 22 min). The cork was then placed in a Petri dish filled with E. coli and was

left overnight to incubate. The next day an inhibition zone would form and the average diameter

would be measured. The (+,+) had the highest average inhibition zone at 57 mm, while the (-,-)

had the lowest inhibition zone at 15 m, this shows much more effective the 12% H2O2 and soaking

time of 22 minutes was over the lower 3% H2O2 and 8-minute soaking time. Both of the variable

effects had a positive effect (26.5 mm and 15.5 mm respectively) meaning that the higher the H2O2

level was and the higher the soaking duration was, the larger the inhibition zone would be. All of

this due to how in 12% H2O2, there is larger amounts of oxidation going on because the solution

has less stabilizers. And because the 22-minute soaking duration allowed for the cork to take in

more of the solution. Both of these allowed the inhibition zone of the (+,+) to be larger than the

others.
 Mojica – Taipalus 23

Other research articles focused mainly on the effect of different concentrations of hydrogen

peroxide and its disinfecting properties. Few articles discussed the length of soaking time for

proper disinfection of E. coli using hydrogen peroxide. None of the research article directly

correlated with the background of the experiment which was to use a solution that would not affect

playability of wooden instrument reeds while maintaining the properties of a disinfectant.

“Commercially available 3% hydrogen peroxide is a stable and effective disinfectant when used

on inanimate surfaces. It has been used in concentrations from 3% to 6% for disinfecting soft

contact lenses” (Rutala and Weber). While the article discusses the everyday uses of hydrogen

peroxide shows that 3% and 6% hydrogen peroxide can be safely used on contact with the human

body, it does not directly correlate with the experiment. The data collected in the experiment

follows data that is similar to the results in other conducted experiments. “Oxidizing agents,

notably hydrogen peroxide (H2O2), are increasingly used in a number of medical, food and

industrial applications but also in environmental ones such as water treatment” (Martin et al.). The

article discusses the uses of hydrogen peroxide and its effectiveness as a non-toxic disinfectant,

which correlates with the goal of the experiment.

Throughout the experiment certain design flaws were found in the procedure that may have

contributed to inaccurate data. One of which being the storage of the H2O2 while the rest of the

experiment was taking place. The H2O2 was kept in open cups near a source of direct sunlight, it

was later understood that the ultraviolet rays from sunlight breakdown the H2O2 and prevent it

from working as well against the bacteria (Li). Another mistake made had to do with the particular

Petri dishes used. The Petri dishes that were used were not as high as expected so in incubation

some of the corks would stick to the top of the Petri dish and not be able to fully spread the H2O2.
 Mojica – Taipalus 24

During the course of the experiment there were a few minor procedural errors made that

could have affected the data. One being that when the corks were being sliced, not all the cuts were

clean. Whether or not it effected the data is unknown, but the unclean cuts did effect the overall

dimensions of the cork used in that trial. Another procedural mistake was that some of the cork,

water, or any other materials could have been contaminated while the experiment took place,

leaving extra bacteria with the E. coli in the Petri dishes. These two were the only documented

mistakes.

Even though the experiment went well and much of the data was useful, more research

should be done on the effects of H2O2 and duration of soaking on E. coli. One suggestion was to

do a three factor DOE and test the effects of light on the H2O2 and how that may or may not interact

with the level of H2O2 used in a given trial. New experiments should also be done to find any other

bacterium that may grow on an instrument reed and what chemicals could safely remove any of

those bacteria while still allowing the safe playability of that reed.

While the 12% hydrogen peroxide and the 22-minute soaking time showed the greatest

zone of inhibition, each hydrogen peroxide concentration (3%, 6%, 12%) is still practically

significant since they all produced a zone of inhibition around the cork. Wooden reed instrument

players will benefit from soaking their reeds in commercially found 3% and 6% hydrogen peroxide

(12% is not widely available at supermarkets/drug stores) because they may notice an increase in

the longevity of their reed because there is a decrease of bacteria on the reed keeping the musician

and reed healthy. Using hydrogen peroxide in place of other sanitizers is also beneficial because

the reed will already be presoaked so that the player will simply remove excess hydrogen peroxide

on the reed and be able to play their instrument. Transporting the reeds in a small container of
 Mojica – Taipalus 25

hydrogen peroxide will keep the reed ready to go. A musician will have the ability to have reeds

ready on the go while staying healthy.

Throughout the course of this lengthy experiment much was learned, not only about the

scientific background behind E. coli and the effect of hydrogen peroxide, but also much more

including; basic scientific procedural concepts, partner work, multitasking and communication,

and most importantly, attention to detail.

 Mojica – Taipalus 26

Works Cited

Cohen, Randy, and Kushner J. Roland. “National Arts Index”. n.p.: n.p., 2013. Web. 9 May

2016. <http://www.artsindexusa.org/wp-content/uploads/2013/09/2013-NAI-Full-

Report.pdf>.

Li, Tyrone. “The Effect of Light on the Decomposition of Hydrogen Peroxide”. n.p.: n.p., 2008.

Web. 11 May 2016. <https://www.scribd.com/doc/3112180/Science-The-Effect-of-Light-

on-the-Decomposition-of-Hydrogen-Peroxide-lab-full-final>.

Linley, Ezra, Denyer P. Stephen, Gerald McDonnell, and Claire Simons. “Use of Hydrogen

Peroxide as a Biocide: New Consderation of its Mechanisms of Biocidal Action”.

n.p.: Journal of Antimicrobial Chemotherapy, 2012. Web. 4 Feb. 2016.

<http://jac.oxfordjournals.org/content/early/2012/04/23/jac.dks129.full.pdf+html>. 

Martin, Nancy L., Bass Paul, and Steven N. . “Antibacterial Properties and Mechanism of

Activity of a Novel Silver-Stabilized Hydrogen Peroxide”. n.p.: PLoS ONE, 2015.

Web. 4 Feb. 2016.

<http://www.plosone.org/article/fetchObject.action?uri=info:doi/10.1371/journal.pone.01

31345&representation=PDF>. 

Rutala, William A., and David J. Weber. “Guideline for Disinfection and Sterilization in

Healthcare Facilities”. n.p.: Center for Disease Control, 2008. 46-47. Web. 14 Mar. 2016.

<http://www.cdc.gov/hicpac/pdf/guidelines/Disinfection_Nov_2008.pdf>.