Abney-Maurice 1

The Effects of Hydrogen Peroxide and Bleach on the Zone of Inhibition in

Relation to Escherichia Coli

Bailey Abney and Jeremy Maurice

Macomb Mathematics Science and Technology Center
Biology 1 9C
Mr. Estapa, Mrs. Dewey, Mr. Acre
May 23, 2013

Abney-Maurice 2
Table of Contents
Introduction .................................................................................................3
Problem Statement .....................................................................................7
Experimental Design ..................................................................................8
Data and Observations .............................................................................20
Data Analysis and Interpretation...............................................................28
Conclusion ................................................................................................39
Acknowledgements ..................................................................................45
Works Cited ..............................................................................................46

Abney-Maurice 3
Introduction
Escherichia coli bacteria are not always as harmful as people tend to
believe; in fact, these bacteria typically cause no harm. An article published by
the Centers for Disease Control states that most Escherichia coli, actually are an
important part of a healthy human intestinal tract, (General). However, there are
some Escherichia coli strains that do cause illness, referred to as pathogenic
strains. Symptoms of infection most commonly include vomiting, severe stomach
cramping, and diarrhea. Strains of Escherichia coli that are considered to be
pathogenic are separated into multiple sub-groups, each with its own name.
Escherichia coli infections are usually caused by infected and undercooked food,
contaminated water, and human and/or animal interaction. There are treatments
for Escherichia coli but in some cases the bacteria can be deadly (General
Information E. coli).
For this experiment, the researchers determined the effect of hydrogen
peroxide and bleach on the zone of inhibition of Escherichia coli. In microbiology,
a zone of inhibition is defined as The clear region around the paper disc
saturated with an antimicrobial agent on the agar surface, (Biology). The clear
region is the area where bacteria do not grow. Hydrogen peroxide and bleach
are both disinfectants, meaning that they should inhibit the growth of the bacteria
and produce a zone of inhibition. Bleach has a specific disinfecting agent called
sodium hypochlorite. In this experiment, the diameters of the zones of inhibition
were measured to the nearest tenth of a centimeter and compared to one
another.

Abney-Maurice 4
The experimenters looked for the greatest zone of inhibition by designing
and completing a two factor DOE composed of three complete runs, each with
seven trials. The factors tested consisted of hydrogen peroxide and Clorox
Bleach. Clorox bleach was specifically used because of the following:
Clorox brand is 5.25% sodium hypochlorite, but many bargain
brands have lower concentrations. The only laundry bleach solution
registered with the EPA as a tuberculocidal disinfectant is Clorox
brand; investigators are encouraged to use Clorox for consistency
in disinfection efficacy (Fact Sheet).
These two factors were tested at varying concentrations by soaking sterilized
paper discs in the equal part solutions. The paper discs were then placed into
petri dishes prepped with agar and Escherichia coli, inverted, and placed in an
incubator set at 37 degrees Celsius overnight. The average zone of inhibition
was measured and used to analyze the effect of hydrogen peroxide and bleach
as well as their interaction with each other.
For this experiment, it was important to know how hydrogen peroxide and
bleach influence bacteria at the cellular level. Hydrogen peroxide is a highly
unstable compound that reacts quickly with many substances. It is commonly
used as a disinfectant because, Contrary to other chemical substances,
hydrogen peroxide does not produce residues or gasses, (Disinfectants).
Hydrogen peroxide breaks down into water and oxygen and the increase of
oxygen in the bacterias growing environment attacks cell proteins therefore
inhibiting the cell in the act of reproduction. Meanwhile, the hydrogen molecules

Abney-Maurice 5
change the pH that bacteria prefer to grow in, making it more acidic. This stunts
growth which, as well as the lack of ability to reproduce, eventually causes death
of the bacteria cells and prevent more bacteria from being produced.
Bleach on the other hand, contains a disinfecting agent known as sodium
hypochlorite. Sodium hypochlorite is often used in the production of disinfectants
because Transport and storage of sodium hypochlorite are safe, (Sodium).
Bleach has been found by the University of Michigan to act upon cells similarly to
the way that high temperatures do. When cells are subjected to especially high
temperatures, they become stressed. This causes the cells proteins to begin
losing their shape and clump together into what are called insoluble aggregates.
This cannot be reversed; therefore, the proteins are damaged and do not work
the way that they should. According to the study, Just like heat, the hypochlorite
in bleach causes proteins to lose their structure and form large aggregates,
(Flannigan). Under this circumstance, the cell is stressed and the proteins are
damaged. Stressed cells eventually die because they are unable to reproduce,
for the proteins are greatly damaged. In addition, the sodium hypochlorite in the
bleach also increases the pH level of the environment that the bacteria grow in,
causing a lack of growth. Both the change of pH level and the damaging of
proteins cause the bacteria to eventually die off.
The results of this experiment will determine what combination of
hydrogen peroxide and bleach is most effective in the act of killing Escherichia
coli. It will also show which factor, hydrogen peroxide or bleach, has a greater
effect on its own. This experiment is beneficial to the community because

Abney-Maurice 6
Escherichia coli are common bacteria and out breaks often spread quickly. It is
important to know how best to disinfect surfaces in the home, in schools, etc. to
help prevent the bacteria from harming people.

Abney-Maurice 7
Problem Statement
Problem:
To determine the effect of hydrogen peroxide and sodium hypochlorite
(bleach) on the zone of inhibition of Escherichia coli (E coli) over a 24 hour
period.
Hypothesis:
The greatest zone of inhibition will result from a combination of a 2.25%
dilution of hydrogen peroxide and 3 drops of bleach per 3.785 liters of distilled
water dilution of bleach.
Data Measured:
The independent variables for this experiment were the concentrations of
hydrogen peroxide and sodium hypochlorite. The dilutions of hydrogen peroxide
were 0.75% as a low, a 1.5% as a standard, and a 2.25% as a high value. The
bleach dilutions were 1 drop of bleach per 3.785 liters of distilled water as a low,
2 drops of bleach per 3.785 liters of distilled water as a standard, and 3 drops of
bleach per 3.785 liters of distilled water as a high. The dependent variable was
the zone of inhibition measured in millimeters and the method of data analysis
was a 2 factor DOE.

Abney-Maurice 8
Experimental Design
Materials:
Escherichia coli sample
180 ml 3% Hydrogen Peroxide,
H202
6 drops 5.25 % Sodium
Hypochlorite, NaOCl, Clorox
(3) 3.785 liter jugs of distilled water
23 g Nutrient agar powder
(21) 8 cm Petri dishes
1L Flask
1000 ml graduated cylinder
100 ml graduated cylinder
(3) 150 ml glass beakers
2L pot
(42) 27 ml Test tubes
1 ml eye dropper
(5) 236.59 ml Mason jars with
lids/rims
Scale (0.01 g precision)
Thermometer (0.1C precision)

Ruler (0.1 cm precision)

24 cm Tongs
16 cm Test tube tongs
20 cm Glass stirring rod
Transfer Loop
12 cm Tweezers
3 cm Stirring magnet
Hot plate
Hot mitt
Incubator, 37C
Towel
Industrial paper towel
Weigh paper
21.59 x 27.94 cm white paper
Electrical tape, 2 cm width
Permanent marker
Hole punch, 7 mm
Ti-Nspire

Sterilization Procedures:
Tong Sterilization
1.

Place the tongs in a 2 liter pot of boiling water for ten minutes without
submerging the handles. This is pictured in Figure 1.

Figure 1. Sterilizing the Tongs

Figure 1 clarifies how to sterilize the tongs without submerging the
handles.

Abney-Maurice 9
Mason Jars, Lids, and Rims Sterilization
2.

Bring a 2 liter pot of water to a boil.

3.

Use sterilized 24 cm tongs to place mason jars, lids, and rims in the pot of
boiling water.

4.

Boil for ten minutes.

5.

Remove from water using the sterile tongs after ten minutes and place on
a towel. Be sure to place the jars upside down to allow excess water to
drain.

6.

Allow to dry and place lids and rims on each jar. Store in a sterile
environment until use.

Transfer Loop Sterilization

7.

Place transfer loop, excluding the handle, in open flame until the metal
turns red directly before use.

Tweezers Sterilization
8.

Place 12 cm tweezers in open flame until the metal turns red directly
before use.

Paper Disc Sterilization

9.

Place the paper discs in a beaker filled with boiling water for 5-10 minutes.

10.

Use 24 cm tongs to remove the beaker from the heat before removing
discs.

Beaker Sterilization
11.

Fill each beaker completely with water and place on a hot plate that has
the temperature control set on high.

12.

Wait for the water to boil. (The time it takes for this to happen will vary
depending on the amount of water in each beaker.)

13.

Once the water boils, allow it to continue boiling for ten minutes before
using 24 cm tongs or a hot mitt to remove the beaker from the hot plate
and empty it of the water.

Abney-Maurice 10
Test Tube Sterilization
14.

Use a pair of 16 cm tongs made for test tubes to dip each test tube in
boiling water several times.

Glass Stirring Rod Sterilization

15.

Place glass stirring rod in boiling water for 5 minutes.

16.

Use a hot mitt to remove from water and place on a paper towel.

17.

Allow to cool before use.

Eye Dropper Sterilization

18.

Fill eye dropper with boiling water and then squeeze the eye dropper to
remove the boiling water after five seconds. Repeat this 3-4 times.

Experimental Procedures:
Bleach Preparation
1.

Label (3) 3.785 liter (equivalent to one gallon) jugs of distilled water using
a piece electrical tape and a permanent marker. Label the jugs High (+),
Standard, and Low (-).

2.

Fill a sterile 1 ml eye dropper with Clorox concentrated bleach.

3.

Add three drops of bleach to the distilled water in the jug labeled High
(+).

4.

Add two drops of bleach to the distilled water in the jug labeled Standard.

5.

Add one drop of bleach to the distilled water in the jug labeled Low (-).

6.

Ensure that the lids are secure on each container and shake each jug
slightly to mix the water with the bleach.

Hydrogen Peroxide Preparation

7.

Label (3) 150 ml sterile glass beakers with a piece of electrical tape and a
permanent marker. One should be labeled High 2.25% (+), one should
be labeled Standard 1.5%, and the last one should be Low 0.75%
(-). This is pictured below in Figure 2.

Abney-Maurice 11
8.

Add 90 ml of hydrogen peroxide and 30 ml of distilled water to the beaker

labeled High -2.25% (+).

9.

Add 60 ml of hydrogen peroxide and 60 ml of distilled water to the beaker

labeled Standard 1.5%.

10.

Add 30 ml of hydrogen peroxide and 90 ml of water to the beaker labeled

Low 0.75% (-).There should be a total of 120 ml of liquid in each
beaker.

11.

Use a sterilized 20 cm glass stirring rod to mix the solution in the beaker
labeled High 2.25% (+). Then rinse and dry the glass stirring rod.

12.

Use the glass stirring rod to mix the solution within the jar labeled
Standard. Then rinse and dry the glass stirring rod.

13.

Use the glass stirring rod to mix the solution within the jar labeled Low
(-). Then rinse and dry the glass stirring rod.

Figure 2. Labeling the Beakers for Diluting Hydrogen Peroxide.

Figure 2 shows how the beakers being used to dilute the hydrogen
peroxide should be labeled High 2.25% (+), Standard 1.5%, and Low
0.75% (-) using tape and a permanent marker.
Soaking Solution Preparation
14.

Place a piece of electrical tape on the lids of (5) sterile 236.59 ml mason
jars. Label them High H2O2, High NaOCl, Low H2O2, Low NaOCl, High
H2o2, Low NaOCl, Low NaOCl, High H2O2, and Standard using a
permanent marker.

15.

Wrap each jar in industrial paper towel so that light will not enter the jars
and the hydrogen peroxide will retain its chemical properties.

Abney-Maurice 12
16.

Place a label on the paper towel wrapping of each jar that matches the
one on the lid. This will prevent the wrong lid from ever being place on the
wrong jar. These matching labels and wrappings are pictured in Figure 3.

17.

Remove the lid from the jar labeled High H2O2, High NaOCl.

18.

Use a 100 ml graduated cylinder to measure and add 20 ml of the High

2.25% (+) hydrogen peroxide solution to the jar labeled High H2O2, High
NaOCl.

19.

Rinse the graduated cylinder.

20.

Use the 100 ml graduated cylinder to measure and add 20 ml of the High
(+) bleach solution to the jar labeled High H2O2, High NaOCl.

21.

Repeat step 19.

22.

Stir using a sterile 20 cm glass stirring rod. Rinse and dry after use.

23.

Screw the lid back onto the jar.

24.

Remove the lid from the jar labeled Low H2O2, Low NaOCl.

25.

Use the 100 ml graduated cylinder to measure and add 20 ml of the Low
0.75% (-) hydrogen peroxide solution to the jar labeled Low H2O2, Low
NaOCl.

26.

Repeat step 19.

27.

Use the 100 ml graduated cylinder to measure and add 20 ml of the Low
(-) bleach solution to the jar labeled Low H2O2, Low NaOCl.

28.

Repeat steps 19, 22, and 23.

29.

Remove the lid from the jar labeled Standard H2O2, Standard NaOCl.

30.

Use the 100 ml graduated cylinder to measure and add 20 ml of the

Standard 1.5% hydrogen peroxide solution to the jar labeled Standard
H2O2, Standard NaOCl.

31.

Repeat step 19.

32.

Use the 100 ml graduated cylinder to measure and add 20 ml of the

Standard bleach solution to the jar labeled Standard H2O2, Standard
NaOCl.

Abney-Maurice 13
33.

Repeat steps 19, 22, and 23.

34.

Remove the lid from the jar labeled High H2O2, Low NaOCl.

35.

Use a 100 ml graduated cylinder to measure and add 20 ml of the High

2.25% (+) hydrogen peroxide solution to the jar labeled High H2O2, Low
NaOCl.

36.

Repeat step 19.

37.

Use the 100 ml graduated cylinder to measure and add 20 ml of the Low
(-) bleach solution to the jar labeled High H2O2, Low NaOCl.

38.

Repeat steps 19, 22, and 23.

39.

Remove the lid from the jar labeled Low H2O2, High NaOCl.

40.

Use the 100 ml graduated cylinder to measure and add 20 ml of the Low
0.75% (-) hydrogen peroxide solution to the jar labeled Low H2O2, High
NaOCl.

41.

Repeat step 19.

42.

Use the 100 ml graduated cylinder to measure and add 20 ml of the High
(+) bleach solution to the jar labeled Low H2O2, High NaOCl.

43.

Repeat steps 19, 22, and 23.

Figure 3. Paper Towel Wrappings and Matching Labels

Figure 3 shows how each jar is wrapped in paper towel and how the label
on the lid matches the label on the paper towel wrapping.

Abney-Maurice 14
Agar Preparation (55-60 Petri Dishes)
44.

Use a 1000 ml flask to measure and add one liter of water to a sterilized
one liter flask.

45.

Place the weigh paper on a scale, and then zero the scale.

46.

Open the bottle of nutrient agar powder and, without placing the lid down,
rotate your wrist to pour the agar powder onto the scale.

47.

Once 23 g of agar powder has been measured, add the powder to the
water in the one liter flask. Avoid getting the powder on the sides of the
flask as best as possible.

48.

Tip the flask with the water slightly, and gently slide a 3 cm magnetic
stirring rod into the flask.

49.

Place the flask onto a hot plate and set the heat level to the highest
setting.

50.

Ensure that the stirring knob is set on 4.

51.

Allow the temperature of the agar to reach 98 degrees Celsius. The

temperature can be checked by placing a thermometer into the flask
(towards the side of the flask to avoid the stirring magnet). Be sure to rinse
the thermometer when finished to remove agar and avoid excess bacteria
growth.

52.

Initially the agar should look like cloudy apple juice, watch for it to change
to the appearance of translucent apple juice.

53.

Once the agar has reached 98 degrees Celsius, use a hot mitt to remove
the flask from the hot plate. Be sure to turn off the hot plate as well.

Petri Dish Preparation

54.

Clam-open one Petri dish at a time to avoid complete exposure to the air.
See Figure 4 below.

55.

Using the hot mitt to hold the flask, pour hot agar into a Petri dish until the
bottom of the Petri dish is covered with agar.

56.

Allow agar to set. This should take about 5-10 minutes. The agar is set
when it turns a very light color and has become a gel-like substance
instead of a liquid.

Abney-Maurice 15
57.

After the agar has set, label the bottom of the petri dish using a permanent
marker with the group name, bacteria type, the date, and the trial type. An
example of this has been shown in Figure 5.

Figure 4. Clamming Open a Petri Dish

Figure 4 above shows how to properly clam open a petri dish before
pouring the agar into it.

Name

Bacteria

Date

Trial Type

Figure 5. Labeling the Petri Dishes

Figure 5 shows an example of how the petri dishes should be labeled with
the group name, the bacteria type, the date, and the trial type. It can be seen
from the picture that this petri dish belonged to the Abney-Maurice group,
contained Escherichia coli, the bacteria would be poured on March 21st, and the
trial was a high-high trial. The petri dish has been labeled towards the edge to
make measuring the zone of inhibition easier later on.

Abney-Maurice 16
Escherichia coli Dilution and Transfer
58.

Add 0.5 ml of distilled water to a sterilized test tube using a sterile 1 ml

eye dropper.

59.

Open the tube of Escherichia coli and drag a sterile transfer loop across
the surface of the bacteria.

60.

Place the transfer loop into the test tube containing 0.5 ml of distilled
water.

61.

Move the transfer loop back and forth in the water to add the Escherichia
coli to the water.

62.

Add 0.5 ml of distilled water to a second test tube.

63.

Sterilize the transfer loop a second time.

64.

Place the sterile transfer loop into the first test tube that already contains
Escherichia coli and distilled water so that a film or bubble of water coats
the loop (like soap on a bubble wand).

65.

Place the transfer loop into the second test tube that contains only distilled
water.

66.

Open a petri dish that has been prepped with agar, and pour the 0.5 ml of
distilled water containing the Escherichia coli from the second test tube
onto the surface of the agar.

67.

Tilt the petri dish back and forth until the liquid covers the entire surface of
the agar.

68.

Replace the lid of the petri dish until ready to apply the paper discs that
have been soaked in various treatments.

Paper Disc Preparation

69.

Use a 7 mm hole punch (does not need to be sterilized) to punch a

minimum of 63 paper discs out of an 21.59 x27.94 cm piece of white
printer paper. Extras should be made in case any paper discs accidently
rip.

70.

Sterilize the paper punches (refer to the paper disc sterilization

procedure).

Abney-Maurice 17
71.

Use sterilized tweezers to remove the paper discs from the beaker once
the paper discs have been sterilized.

72.

Place on a sheet of industrial paper towel and allow to dry before soaking.

Randomization of the Trials

73.

From the home screen of the Ti-Nspire calculator, choose the calculator
option.

74.

Press the menu button and select Probability.

75.

Select Random from the sub-menu and then select Integer.

76.

Enter the range of the integers to be used with a comma in between. For
this experiment 1, 7 should be entered.

77.

Hit Enter to generate random integers.

78.

Fill in the Trial Order column of a blank DOE data table without repeating
any numbers and leaving runs one, four, and seven as standard trials.

Testing the Factors

79.

Using sterilized 12 cm tweezers, add three dry, sterilized paper discs to

each soaking solution except for the Standard H2O2, Standard NaOCl
solution. Instead add nine paper discs to the standard solution.

80.

Allow the discs to soak for a minimum of five minutes.

81.

Use the sterilized tweezers to remove the discs paper discs from the
solution associated with the first trial and gently press them into a petri
dish that has been prepped with agar and coated with Escherichia coli.
The solution that the discs were soaked in should correspond with the trial
type already labeled on the petri dish. The discs should be equally
spaced. This has been pictured in Figure 6.

82.

Invert the petri dish and place in the incubator set at a temperature of 37
degrees Celsius.

83.

Repeat steps 81 and 82 for the remaining six trials. The order will of
the trial types will depend on the random integer function of the Ti-Nspire
calculator.

84.

After 24 hours in the incubator, calculate the overall zone of inhibition for
each petri dish (see below for details).

Abney-Maurice 18

85.

Record the average zone of inhibition for each trial in the result column of
a DOE table.

86.

Repeat steps 84 and 85 for each petri dish.

87.

Repeat steps 79 through 86 for two additional runs.

Figure 6. Spacing the Paper Discs

Figure 6 shows how the paper discs should be spaced in the petri dish.
Measuring the Zone of Inhibition
88.

Draw the zone of inhibition (the area around each paper disc where
bacteria do not grow) with a permanent marker.

89.

Draw two lines through each paper disc that resemble a crosshair. This is
pictured in Figure 7.

90.

Measure the distance of each line then add the measurements together
and divide by two to get the average zone of inhibition for each disc.

91.

Repeat steps two and three for the remaining two discs in the petri dish.

92.

Add the three average zones of inhibition for the petri dish (one for each
disc) and divide by three to find the average zone of inhibition of the
petri dish as a whole.

Abney-Maurice 19
Crosshairs

Zones

Figure 7. Drawing the Zone of Inhibition

Figure 7 shows how the zone of inhibition was drawn for one trial as well
as the crosshairs used to measure it.

Abney-Maurice 20
Data and Observations
Data:
Table 1
Factors Tested in Experiment
Factors
Hydrogen Peroxide (%)
(-) Low

0.75

Standard

1.5

Sodium Hypochlorite/Bleach (drops/3.785 L)

(+) High

2.25

(-) Low

Standard

(+) High

Table 1 above shows the low, standard, and high values at which
hydrogen peroxide and bleach were tested during the experiment. The standard
values were determined by researching the level of each substance that would
kill the bacteria and then lowering that value so that a zone of inhibition could be
seen. These standard values were then raised slightly and lowered slightly to
create the low and high values of each substance. For hydrogen peroxide, the
standard value was raised or lowered by 0.75% to find the low and high testing
values; for bleach the standard value was raised or lowered by 1 drop to find the
low and high testing values.

Abney-Maurice 21
Table 2
DOE Trial Order, Type, and Results
Run 1
Trial
Order
1

Trial
Type
Standar
d

(+,+)

(-,-)

Standar
d

(+.-)

(-,+)

Standar
d

Run 2
Result
(cm)
3.1
3.3
2.7
3.4
2.7
3.9
5.0

Trial
Order
1

Trial
Type
Standar
d

(+,+)

(-,-)

Standar
d

(+.-)

(-,+)

Standar
d

Run 3
Result
(cm)
3.5
4.4
1.5
2.4
3.0
4.8
4.9

Trial
Order
1

Trial
Type
Standar
d

(+,+)

(-,-)

Standar
d

(+.-)

(-,+)

Standar
d

Resul
t (cm)
4.7
4.2
0.3
3.2
2.5
1.8
2.0

Table 2 shows the order that the trials were run in during the experiment
and the result of each trial. The table also shows that the DOE consisted of three
runs, each of which contained seven trials.

Abney-Maurice 22
Observations:
Table 3
Run 1 Observations
Run 1
Date
3/18/2013

Observations
Run 1 Prep: Solutions made on 3/11/13, all plates were
poured on 3/18/13, discs were boiled and soaked on
3/18/13
Trial 1: (Standard 1) Very large zones with relatively small
colonies
Trial 2: (+,+) Two of the discs were pushed together
creating only zone of inhibition, two large zones and
bacteria found around the edge of the plate
Trial 3: (-,-) Extremely small zones, almost entirely bacteria,
random zone of inhibition present that does not encompass
a disc

3/19/13, 3/20/13

Trial 4: (Standard 2) Larger colonies around the edges,

oddly shaped zones of inhibition
Trial 5: (-,+) Original dish showed no zone of inhibition, trial
was redone on 3/19/20 and the zone of inhibition was remeasured on 3/20/13, New trial showed one zone smaller
than other two, smaller colonies in the middle of the plate
Trial 6 : (+,-) One disc has no zone of inhibition, other two
discs had roughly equal zones, bacteria around the edges
of the plate
Trial 7: (Standard 3) One disc had no zone of inhibition,
other two discs had oddly shaped zones with large colonies

Table 3 shows the observations made during run one of the DOE. The
observations have been organized by date and labeled either Prep or Trial _
to indicate whether the prep of the trials or a specific trial is being discussed. It
can also be seen from the table what order the trials occurred in by looking at the
test within the parenthesis next to each trial number.

Abney-Maurice 23
Table 4
Run 2 Observations
Run 2
Date:

3/20/2013

Observations
Run 1 Prep: Three plates poured March 12, 2013 [Standard; (+), (+);
(-), (-)] and were stored in the refrigerator; remaining plates were
poured on March 18, 2013 [Standard 2; Standard 3; (+), (-); (-), (+)].
Standard solution was remade due to leaking and air exposure.
Experimenters noticed that the E coli spread easier on the warm
plates compared to the cold plates.
Trial 1: (Standard 1) One zone of inhibition larger, other two zones
somewhat symmetric
Trial 2: (+,-) Very large zones of inhibition that overlap, few large
bacteria colonies
Trial 3: (-,-) Two zones of inhibition roughly symmetric, third zone is
slightly bigger, small bacteria colonies

3/21/2013

Trial 4: (Standard 2) Oddly shaped symmetric zones of inhibition with

large bacteria colonies
Trial 5: (+,+) Zones of inhibition not symmetric, but about the same
size, one disc may have moved
Trial 6 : (-,+) Bacteria looks watery, no zones of inhibition
Trial 7: Some large bacteria colonies, zones of inhibition different
sizes, but largest zones roughly symmetric

Table 4 shows the observations made during run two of the DOE. The
observations have been organized by date and labeled either Prep or Trial _
to indicate whether the prep of the trials or a specific trial is being discussed. It
can also be seen from the table what order the trials occurred in by looking at the
test within the parenthesis next to each trial number.

Abney-Maurice 24
Table 5
Run 3 Observations
Run 3
Date
3/21/2013

Observations
Run 3 Prep: Discs for run three were boiled and soaked on March
20, 2013; plates were poured on 3/20/13
Trial 1: (Standard 1) One large bacteria colony, no other growth,
large zones of inhibition
Trial 2: (-,+) Zones of inhibition toward edge of the plate, medium
sized bacteria colonies toward the middle of the plate
Trial 3: (+,-) Large zones of inhibition with a few small bacteria
colonies

3/22/2013

Trial 4: (Standard 2) Lots of bacteria growth, two similar sized

zones of inhibition and third zone smaller
Trial 5: (-,-) Lots of bacteria growth, two very small zones of
inhibition, third zone larger
Trial 6 : (+,+) Zones of inhibition very large, about the same size,
bacteria growth in one spot
Trial 7: (Standard 3) Zones of inhibition same size, large colonies
of bacteria toward the outer edge of the plate

Table 5 shows the observations made during the third run of the DOE.
The observations have been organized by date and labeled either Prep or Trial
_ to indicate whether the prep of the trials or a specific trial is being discussed. It
can also be seen from the table what order the trials occurred in by looking at the
test within the parenthesis next to each trial number.

Abney-Maurice 25

Figure 8. Run 1 Petri Dishes

Figure 8 above shows images of petri dishes from Run 1 of the DOE after
they were placed in the incubator at 37 C for 24 hours. Each image is labeled
according to its trial type and the order that the trials occurred. The concentration
of bacteria colonies has resulted from different combinations and values of
hydrogen peroxide and bleach. It can be seen from the figure that an obvious
error had occurred with the (-), (+) trial because a zone of inhibition did not form
around any of the paper discs; therefore, it was redone. The figure also shows
that the (+), (-) trial had the greatest zone of inhibition and the (-), (+) had the
smallest zone of inhibition when the original (-), (+) trial is not considered.

Abney-Maurice 26

Figure 9. Run 2 Petri Dishes

Figure 9 above shows images of petri dishes from run two of the DOE
after they were placed in the incubator at 37 C for 24 hours. Each image is
labeled according to its trial type and the order that the trials were run in. The
concentration of bacteria colonies has resulted from different combinations and
values of hydrogen peroxid3e and bleach. The figure shows that Standard 3 and
(+), (-) trials had the largest zones of inhibition while the (-), (-) trial had the
smallest zone of inhibition.

Abney-Maurice 27

Figure 10. Run 3 Petri Dishes

Figure 10 above shows images of petri dishes from run 3 of the DOE after
they were placed in the incubator at 37 C for 24 hours. Each image is labeled
according to its trial type and the order that the trials were run in. The
concentration of bacteria colonies has resulted from different combinations and
values of hydrogen peroxide and bleach. It can be seen from the figure that the
Standard 1 trial had the greatest zone of inhibition while the (-), (-) trial had the
smallest zone of inhibition for run three.

Abney-Maurice 28
Data and Analysis and Interpretation
Data Analysis:
Table 6
DOE Results and Averages
Runs
Run One
Hydrogen
(cm)
Bleach
Peroxide
+
+
3.3
2.7
+
2.7
+
3.9

Run 2
(cm)

Run 3
(cm)

Average
(cm)

4.4
1.5
3.0
4.8

4.2
0.3
2.5
1.8

4.0
1.5
3.5
2.7
Grand
Average:
2.9

Table 6 shows the average zone of inhibition that was found for each
combination of hydrogen peroxide and bleach, excluding the standard trials, for
each run of the DOE. The average zone of inhibition of each combination for the
complete DOE (all three runs) is also given. The grand average is shown by the
table as well, which is the average zone of inhibition of all four combinations for
all three runs. These numbers have all been rounded to the nearest tenth but
kept exact for later calculations.

Abney-Maurice 29
Table 7
Effect of Hydrogen Peroxide
Hydrogen Peroxide
(-) Amount
(0.75%)

(+) Amount
(2.25%)

1.5

4.0

2.7

3.5

Average= 2.10

Average= 3.75

Table 7 shows the average zone of inhibition, in centimeters and rounded

to the nearest tenth, when hydrogen peroxide was held low and when hydrogen
peroxide was held high. The exact versions of these numbers were used to find
the average of each column. These averages were rounded to the nearest
hundredth. The table shows that when hydrogen peroxide was held low the zone
of inhibition was 2.10 centimeters on average. The table also shows that the
zone of inhibition was 3.75 centimeters on average when hydrogen peroxide was
held high. These averages can then be used to find the effect of mass by
subtracting the low average from the high average. Once calculated, the effect
on hydrogen peroxide is found to be 1.65 when rounded to the nearest

Zone of
Inhibition (cm)

hundredth.

2.10

-2

-1

5
4
3
2
1
0

3.75

Hydrogen Peroxide

Figure 11. Effect of Hydrogen Peroxide

Abney-Maurice 30
Figure 11 above shows that the effect of hydrogen peroxide is 1.65. This
effect value has been rounded to the nearest hundredth and shows that as the
amount of hydrogen peroxide in the solution increases, the zone of inhibition
increases by 1.65 centimeters.
Table 8
Effect of Bleach
Bleach
(-) Amount
(ml)

(+) Amount
(ml)

1.5

4.0

3.5

2.7

Average= 2.50

Average= 3.35

Table 8 shows the average zone of inhibition in centimeters, rounded to

the nearest tenth, when bleach was held low and when bleach was held high.
Each column was then averaged by using the exact averages. These numbers
were then rounded to the nearest hundredth. The table shows that when bleach
was held low the zone of inhibition was 2.50 centimeters on average. The table
also shows that the zone of inhibition was 3.35 centimeters on average when
bleach was held high. These averages can then be used to find the effect of
mass by subtracting the low average from the high average. Once calculated, the
effect of bleach is found to be 0.85 when rounded to the nearest hundredth.

Abney-Maurice 31

Zone of Inhibition
(cm)

5
3.35

4
2.50

3
2
1
0

-2

-1

Bleach
Figure 12. Effect of Bleach
Figure 12 above shows that the effect of bleach is 0.85. This effect value
has been rounded to the nearest hundredth and shows that as the amount of
bleach in the solution increases, the zone of inhibition increases by 0.85
centimeters.
Table 9
Interaction Effect of Hydrogen Peroxide and Bleach

Hydrogen
Peroxide

Bleach
(-) Amt
(+) Amt
(ml)
(ml)
Solid
Segment

(+) Amt
(2.25%)

3.5

Dotted
Segment

(-) Amt
(0.75%)

1.5

2.7

Table 9 shows the interaction of hydrogen peroxide and bleach on the

average zone of inhibition. The values given are the averages of all three runs of
the DOE and have been rounded to the nearest tenth.

Abney-Maurice 32

Zone of Inhibition
(cm)

5
3.5

4.0

4
2.7

3
1.5

Hydrogen
Peroxide (-)

Hydrogen
Peroxide (+)

1
0
-2

-1

Bleach

Figure 13. Interaction of Bleach and Hydrogen Peroxide

Figure 13 above shows the interaction of hydrogen peroxide and bleach.
The solid line segment represents when hydrogen peroxide was held high while
the dotted line segment represents when hydrogen peroxide was held low. The
x-axis then shows whether bleach was being held low or high. The values on the
graph are rounded to the nearest tenth but were kept exact when calculating the
interaction effect. The interaction effect was found to be 0.35. Because the line
segments are not parallel, it is suggested that the interaction of the two factors
affected the zone of inhibition.

Abney-Maurice 33

Value of Stnadard (cm)

10
8
5.0

3.5

3.1 3.4

4.9 4.7
3.2

2.4

Standard

2.0

2
0
0

10

Standard Number

Figure 14. Standards Scatter Plot

Figure 14 shows a scatter plot of the results of the standard trials. The
scatter plot shows that the zones on inhibition for the standard trials, rounded to
the nearest tenth, ranged from two centimeters to five centimeters. The spacing
of the data shows that there may have been an error during the experiment
because the data is not very consistent.

Figure 15. Dot Plot of Effects

Figure 15 above shows the effect of hydrogen peroxide (HP), the effect of
bleach (B), and the effect of hydrogen peroxide and bleach (HPB). When
multiplying the range of standards (3) by two it is found that significant factors
should lie outside the range of

Abney-Maurice 34
-6 to 6 on a number line. The dot plot shows that none of the factors were found
to be significant, meaning that error may have occurred during the experimental
design and/or during the actual experiment.
Interpretation:
Figure 11 shows a graph of the effect of the first variable, hydrogen
peroxide. To determine the effect, the average zone of inhibition for each trial
type for all three runs of the DOE had to be found. These averages are shown by
Table 6. Then, the effect was calculated by averaging the zones of inhibition
when hydrogen peroxide was held low and by averaging the zones of inhibition
when hydrogen peroxide was held high. This is shown by Table 7. The average
zone of inhibition when hydrogen peroxide was held low was found to be 2.10
centimeters and the average zone of inhibition when hydrogen peroxide was held
high was found to be 3.75 centimeters, also shown by Table 7. The low average
(2.10 centimeters) was then subtracted from the high average (3.75 centimeters)
to determine the numerical effect of hydrogen peroxide. When looking at the
graph, it can be seen that the line has a positive slope and that the effect of
hydrogen peroxide came out to be 1.65. This means that as the level of hydrogen
peroxide in the solution increased from low to high, the zone of inhibition
increased by 1.65 centimeters. Since hydrogen peroxide is meant to be a
disinfectant, the increase of the area that the bacteria does not grow (zone of
inhibition) shows that hydrogen peroxide was effective.
The effect of bleach was calculated in the same way but with different
data. Again the average zones for all three runs of the DOE that are shown in

Abney-Maurice 35
Table 6 were used to find the average zone of inhibition when bleach was held
low and when bleach was held high. Table 8 shows that the average zone of
inhibition when bleach was held low was found to be 2.50 centimeters and the
average zone of inhibition when bleach was held high was found to be 3.35
centimeters. The low average was then subtracted from the high average to
determine the numerical effect that bleach had on the zone of inhibition. The
effect of bleach was found to be 0.85. A graphical representation of the effect of
bleach is shown by Figure 12. This means that as the bleach level in the solution
increased from low to high the zone of inhibition increased by 0.85 centimeters.
Bleach is also intended to act as a disinfectant; therefore, the increase of zone of
inhibition shows that it killed bacteria as it was meant to do. When compared to
the effect of hydrogen peroxide, it can be seen that both factors did what they
were intended to do increase the area around a treated paper disc where
bacteria did not grow. The effects were very similar; however, hydrogen peroxide
did have a greater effect overall.
Table 9 shows the interaction between hydrogen peroxide and bleach.
The values given in the table are the average zones of inhibition for all three runs
of the DOE. When reading the table, the average zone of inhibition for the highhigh, high-low, low-high, and low-low trials can be seen. These numbers are then
represented by a line graph in Figure 13. In Figure 13, the dashed line segment
represents when hydrogen peroxide was held low and the solid line segment
represents when hydrogen peroxide was held high. Points that are plotted in
alignment with negative one on the x-axis represent when bleach was held low.

Abney-Maurice 36
The plotted points that align with positive one on the x-axis represent just the
opposite, when bleach was held high. The graph shows that the two lines are
nearly parallel; however, when the slope of the dashed line segment was
subtracted from the slope of the solid line segment, a difference of -0.35 was
found. This could mean that at some values of hydrogen peroxide and bleach
other than the ones that were tested, may have some sort of significant
interaction. However, since the lines do not intersect there is most likely no
significant interaction. The slight interaction can be seen though. When bleach
was analyzed on its own, it was predicted that the zone of inhibition should be
about 2.50 centimeters when bleach was held low. But by looking at the
interaction graph it can be seen that when a low value of bleach was combined
with a low value of hydrogen peroxide the zone of inhibition was lower than
expected at 1.5 centimeters. This means that when low hydrogen peroxide was
added to the low amount of bleach the effect was inhibited. The same result
occurred when a low value of hydrogen peroxide was combined with a high
amount of bleach. When bleach was analyzed on its own it was found that the
zone of inhibition was about 3.35 centimeters when the amount of bleach in the
treatment solution was high. However, when the high value of bleach was
combined with the low value of hydrogen peroxide the zone of inhibition was
again decreased, this time to about 2.7 centimeters. This shows that when the
solution had a low amount of hydrogen peroxide and either a low or high amount
of bleach, the zone of inhibition decreased by a rather large amount. On the
other end, when a high amount of hydrogen peroxide was combined with a low

Abney-Maurice 37
amount of bleach the zone of inhibition actually increased. A low value of bleach
produced a 2.50 centimeter zone of inhibition on average, but when combined
with a high amount of hydrogen peroxide the zone of inhibition that resulted
measured 3.50 centimeters. This increase of area where no bacteria grew
occurred again when a high amount of bleach was combined with a high amount
of hydrogen peroxide. Bleach, when tested at a high value, created a 3.35
centimeter zone of inhibition but the high-high combination of bleach and
hydrogen peroxide produced a zone of inhibition measuring 4.0 centimeters. This
shows that a high value of hydrogen peroxide in the treatment enhanced the
ability of the bleach to kill the bacteria. This shows that bleach, of a high or low
value, works best in combination with a high value of hydrogen peroxide and is
repressed when combined with a low value of hydrogen peroxide.
Figure 14 shows a scatter plot of the standard trial results rounded to the
nearest tenth of a centimeter. The graph shows a lack of pattern with the
exception of a slight decreasing trend at the far right of the graph. There is a
range of three for the range of standards and the lack of consistency shows that
there were more than likely errors in the experimental design and/or the
execution of the experiment itself. The range of standards was used to determine
the statistically significant factors. To do so, the range of standards was
multiplied by two and was then used to find a range of values that would
determine the factors to be either significant or insignificant. Since the range of
standards multiplied by two equals six, a factors effect value had to be above
positive six or below negative six to be deemed significant. Figure 15 shows that

Abney-Maurice 38
the effect values of hydrogen peroxide, bleach, and their interaction all fell within
the range of negative six to positive six. This means that none of the factors
tested during the experiment were significant. The experimenters were not
surprised by the lack of statistical significance because the range of standards
was three centimeters and the range of the entire data set was approximately
three centimeters. The results suggest that errors had occurred during the design
and/or implementation of the experiment because the experiment was designed
such that something should have been statistically significant. However,
hydrogen peroxide did have the greatest effect value even though it was not
statistically significant. This suggests that if the experiment was redone,
hydrogen peroxide may be determined to be statistically significant factor. This is
supported by the previous paragraph that discusses how the amount of hydrogen
peroxide in the treatment determined whether bleach worked better or worse
than it would have on its own.

Abney-Maurice 39
Conclusion
The results of the experiment indicated that the when a solution containing
a high amount of hydrogen peroxide (a 2.25% solution) and a high amount of
beach (a three drops per 3.785 liters of distilled water solution), the greatest
average zone of inhibition was found; therefore, the experimenters hypothesis
was accepted. This experiment was intended to find what combinations of
hydrogen peroxide and bleach would most greatly inhibit the growth of the
bacteria Escherichia coli. A 2-factor DOE was designed and utilized to find the
answer to this.
The experiment showed that the solutions containing low amounts of
hydrogen peroxide combined with bleach allowed more bacteria to grow than
would be expected if bleach had been the lone disinfectant at work. This was
found to hold true whether the low amount of hydrogen peroxide was combined
with a low amount of bleach or a high amount of bleach. For example, it was
expected that when bleach was held low on its own that the average zone of
inhibition produced would be 2.50 centimeters, but when a low level of bleach
was combined with a low level of hydrogen peroxide, the average zone of
inhibition produced was only 1.50 centimeters. It was also found that the
solutions containing high amounts of hydrogen peroxide combined with bleach
killed more bacteria than what would have been expected if bleach was the only
disinfectant being utilized. This happened when bleach was held low and when
bleach was held high. Again, the average zone of inhibition expected to be
produced when bleach was held low on its own was 2.50 centimeters; however,

Abney-Maurice 40
when a low level of bleach was combined with a high level of hydrogen peroxide,
the average zone of inhibition that resulted was 2.5 centimeters. This means that
low amounts of hydrogen peroxide inhibited the bleachs ability to disinfect while
high amounts of hydrogen peroxide were advantageous.
Once again, the hypothesis made by the experimenters before the start of
the experiment was accepted, for the high-high combination of hydrogen
peroxide and bleach was found to produce the greatest zone of inhibition during
the experiment. These results occurred because the high-high combination of
hydrogen peroxide and bleach likely attacked the cell proteins, rendering the
cells unable to reproduce, and increased the pH level more than the other
combinations of hydrogen peroxide and bleach. An analysis of the DOE showed
that hydrogen peroxide, bleach, nor their interaction was found to have statistical
significance. This does not mean that they did not affect the bacteria at all; the
experiment clearly showed that the factors did affect the Escherichia coli and
also had an effect on each other. Instead, the lack of statistical significance
points in the direction of experimental flaws that occurred, which likely affected
the results of the experiment.
The lack of statistical significance of the factors showed that there were
flaws present in the design of the experiment. One of these flaws was that the
sterilization of the petri dishes was overlooked. This means that bacteria other
than the Escherichia coli that was transferred into the petri dishes by the
experimenters was likely present and would have affected the results of the
experiment. There was also no way of sterilizing the paper towel that the paper

Abney-Maurice 41
discs were set on in order for them to dry before being soaked in the various
solutions. There may have been bacteria present on the paper towel that
contaminated the paper discs before they were soaked and placed in the petri
dishes. This too would have affected the experimental results. Another possible
flaw is that the solutions were not remade each day, meaning that they could
have been contaminated or lost some of their potency (especially since hydrogen
peroxide is such an unstable compound) over the course of the experiment. Also,
due to the size of the petri dishes, the zones of inhibition sometimes overlapped.
This made it hard to measure each zone and could have altered the results of the
experiment. The experimenters attempted to compensate for this by using larger
petri dishes after this complication was found during pre-trials, but the zones still
overlapped. Ideally, the petri dishes would have been large enough to show the
zones of inhibition in their entirety.
Errors also took place during the execution of the experiment. First, the
agar that was used during the experiment was an issue. The agar was not made
by the same person every day and was shared between several groups of
experimenters. This means that the agar may have varied slightly from run to run
and may have been contaminated by factors used by other groups. Another
possible error is that all of the petri dishes were not prepped with agar on the
same day. The experimenters intended to prep petri dishes with agar the day
before a run to save time and the prepped dishes were stored in the refrigerator.
However, sometimes problems arose where petri dishes had to be used and
more had to be poured on the day of a run. The lack of consistently of the date

Abney-Maurice 42
that the agar was poured and the temperature of the agar when the Escherichia
coli was transferred to the prepped petri dishes may have also affected the
results of the experiment.
The paper discs soaked in the various solutions also posed problems.
Some of the discs moved after being positioned in the petri dishes when the petri
dishes were inverted before being placed in the incubator. This would have
caused the solution to cover a greater area than that of the paper disc resulting in
a greater zone of inhibition. Another error is that the discs for each trial were not
soaked in the solutions for the exact same amount of time due to time restraints
of the experiment. The fact that the same experimenter did not always preform
the same tasks during the runs of the experiment, which could have caused
small changes in the way things were executed, was yet another problem. All of
these errors likely contributed to the results of the experiment including the lack
of statically significant factors.
If further research were to be conducted the experiment itself could be
repeated with several more runs and corrected errors in order to collect more
accurate data. The experiments variables could also be changed to different
levels to see where a significant interaction between the two factors occurred.
The same could be done to see at what level a lone factor was statistically
significant. Several similar experiments could also be conducted using the same
variables at several different levels to see the minimum amount of the factors
needed to kill the Escherichia coli entirely. This research project showed that low
amounts of hydrogen peroxide inhibited the bleachs ability to produce a zone of

Abney-Maurice 43
inhibition and that high amounts of hydrogen peroxide increased the ability of
bleach to produce a zone of inhibition. These findings could also be expanded
upon by conducting a different experiment(s) using hydrogen peroxide and
another common disinfectant other than bleach to see if hydrogen peroxide
affects other disinfectants the way that it affects bleach. Hydrogen peroxide was
also found to have a greater effect on Escherichia coli than bleach. If other
factors were tested along with hydrogen peroxide, it could be determined what
disinfectants, if any, work more efficiently than hydrogen peroxide.
The general public will benefit from this research because the findings can
better protect people from pathogenic strains of Escherichia coli. Potentially
contaminated surfaces can be more effectively disinfected by knowing the effect
values of hydrogen peroxide, bleach, and their interaction. The information
gathered by the experiment allows the appropriate levels of the disinfectants to
be used and for potentially contaminated surfaces to be more efficiently
disinfected. The experiment showed that hydrogen peroxide has a greater effect
than bleach as well as the interaction between the two factors. This means that
hydrogen peroxide should be used to disinfect where Escherichia coli is
concerned. More efficiently disinfected surfaces can prevent illness from
occurring. This is especially important for places such as schools and residences
for the elderly because children and the elderly are the most susceptible to
infection. This will not only prevent illness that will need to be treated but also
death caused by pathogenic strains of Escherichia coli.

Abney-Maurice 44
From this experiment, the experimenters learned the importance of
thorough planning during the designing of an experiment. They also learned the
importance of sterilization. It was found that time restraints tended to cause
errors during the execution of the experiment. Therefore in future experiments, it
could be beneficial to the experimenters to design a less complex experiment.

Abney-Maurice 45
Acknowledgement
The experimenters would like to acknowledge the people who helped
make their research possible. These people include Mr. Estapa, Mr. Acre, Mrs.
Hilliard, and Mrs. Dewey. Mr. Estapa taught the experimenters how to work with
the Escherichia coli and also helped to plan the experiment. Mr. Acre was of
much help during the analysis and interpretation of the data that was collected.
He also helped the experimenters analyze what possible errors could have been
made during the experimental design as well as the execution of the experiment
that may have led to the lack of statistically significant factors. Mrs. Hilliard and
Mrs. Dewey played a large role in teaching the experimenters how to format and
write a formal research paper for the first time. Mrs. Hilliard was especially helpful
to the experimenters in the areas of organization and refinement of the
experimental design while Mrs. Dewey was especially helpful in the aspect of
guiding the experimenters during the writing of the introduction and conclusion.
All of these people gave the experimenters the knowledge and guidance that
they needed in order to create, conduct, and complete their research.

Abney-Maurice 46
Works Cited
"Disinfectants Hydrogen Peroxide." Hydrogen Peroxide as a Disinfectant.
Lenntech Water Treatment Solutions, n.d. Web. 14 May 2013.
<http://www.lenntech.com/processes/disinfection/chemical/disinfectantshydrogen-peroxide.htm>
"Disinfectants Sodium Hypochlorite." Sodium Hypochlorite as a Disinfectant.
N.p., n.d. Web. 12 May 2013
<http://www.lenntech.com/processes/disinfection/chemical/disinfectantssodium-hypochlorite.htm>
"General Information Escherichia Coli (E. Coli)." Centers for Disease Control and
Prevention. Centers for Disease Control and Prevention, 03 Aug. 2012.
Web. 12 May 2013. < http://www.cdc.gov/ecoli/general/#symptoms>
"Zone of Inhibition." - Definition from Biology-Online.org. N.p., 22 June
2008. Web. 12 May 2013.
< http://www.biology-online.org/dictionary/Zone_of_inhibition>
Ross-Flanigan, Nancy. "Clean Results: U-M Researchers Learn How Bleach Kills
Bacteria." Http://www.ur.umich.edu/0910/Jan11_10. University of
Michigan, n.d. Web. 27 Feb. 2013.
<http://www.ur.umich.edu/0809/Nov17_08/13.php>.
Oregon State University. Disinfection Using Chlorine Bleach. Corvallis: Oregon
State University, 2011. Http://oregonstate.edu/. Oregon State University.
Web. 28 Feb. 2013.

Abney-Maurice 47
<http://oregonstate.edu/dept/larc/sites/default/files/pdf/chlorine-factsheet.pdf>.