Cherry McMain 1

Introduction
There are many different types of hand sanitizers. They come in different
sizes, and more importantly concentration percentages of ethyl alcohol. When
the consumer goes to the store, they may see the more expensive hand
sanitizers, and the off brand, less expensive hand sanitizers. Which is more likely
to be bought? Logically the less expensive will be bought, but is it worth it.
The main difference between the off brand and the main brand hand
sanitizers is the concentration percentage of ethyl alcohol, or C2H6O (Chu). For
example, Kroger Brand Hand Sanitizer has 62% ethyl alcohol concentration,
whereas Purell Brand Hand Sanitizer has 70%. (This experiment aimed to see
how important the percentage of ethyl alcohol concentration has to the colony of
Escherichia-coli.) Colonies of Escherichia-coli were exposed to three different
concentrations of ethyl alcohol and put in different temperature incubators, to see
how many colonies of Escherichia-coli would grow. By analyzing the data for
each factor, (temperature and ethyl alcohol) and their interaction, the overall
effect of temperature and ethyl alcohol on colonies of Escherichia-coli will be
found.
In one similar experiment, bacteria was only exposed to very low
concentrations of ethyl alcohol (Chatterjee) and it was found that bacteria can
grow in low concentration ethyl alcohol, but as the concentration increased,
Escherichia-coli is harder to grow. In another experiment, Escherichia-coli
plasma membrane was exposed to ethyl alcohol (Dombek). As ethyl alcohol

Cherry McMain 2

concentration went up, the fluidity of the plasma membrane increased as well. A
final experiment had different test subjects put their hands into colonies of
Escherichia-coli. Different treatments were given to see which the most efficient
(Reynolds) was.
This is an important topic because the importance of hand sanitizer has
been so commercialized, and it is good for the consumer to know what the most
efficient hand sanitizer there is. Because sicknesses go around so much, it would
be best for everyone to know what the best kind of hand sanitizer to use.
Although, if there is excessive use of hand sanitizer, there is the possibility of
getting acute pancreatitis, which is the sudden inflammation of the pancreas
(Huynh-Delerme).

Cherry McMain 3

Problem Statement:
Problem:
What will happen if Escherichia-coli is grown in petri dishes at 36C, 37C
and 38C along with putting Ethyl Alcohol Soaked disks in the middle if the
dishes at 68%, 69% and 70% to see how many bacteria colonies will grow close
to the Ethyl Alcohol soaked disks.
Hypothesis:
If colonies of Escherichia-coli are exposed to 68% ethanol and are placed
in a 38 C incubator, then the most colonies of Escherichia-coli will grow.
Data Measured:
The two independent variables we have are temperature and percent of
Ethyl Alcohol. Temperature is measured in degrees Celsius. The percent of Ethyl
Alcohol is measured in percent per ml. The dependent variable is the distance EColi is away from the ethanol soaked disk. That will be measured in mm. The
concentration of 68, 69, and 70% were chosen because 70% Ethyl Alcohol is the
amount in Purell Hand Sanitizer, and 68% and 69% would give enough
difference to show if more ethyl alcohol is more efficient than less. The standard
of 37C was chosen because that is the average human body temperature. The
low of 36C and the high of 38C were chosen because they could give enough
difference to see if hotter or colder temperatures affect efficiency.

Cherry McMain 4

Experimental Design
Materials:
Bunson Burner
24 cm Tongs
Escherchia coli
Distilled Water
10 ml Graduated Cylinder
200 ml Ethyl Alcohol
Transfer Loop
1 ml Eye Dropper
Hot Pad
10 cm Stirring Magnet

Incubator (36, 37, 38 C)

40 Petri Dishes 5 x 1.5 cm
8 mm Paper Disks/Hole Punches
23 g Agar Nutrient
1 L Flask Weigh Boat
TI nSpire Calculator
Ten 10 ml Test Tubes
Full Match Book
Black Sharpie
Paper

Agar Prep:
1
2
3
4
5
6
7
8
9

Measure water in a one liter flask.

Measure 23 grams of nutrient agar on an electric scale.
Place the 23 grams into the flask of water.
Slide a stirring magnet into the flask.
Place the flask on a hot plate at the hottest level.
Turn the stirring knob to four.
Wait for the mixture to go from cloudy to clear looking.
Use a hot mitt to grab the flask off the hot plate.
Clam petri dishes open.
10 Pour the mixture into each separate petri dish until it covers the
bottom.
11 Wait for the mixture to become solid.
12 This should make enough for the entire experiment.

Bacteria Prep:
1 Put around 50 ml of water into a 75 ml Beaker and boil it on a hot plate.
2 Place ten test tubes into the boiling water for around one minute to
sterilize them.
3 Light Bunsen burner with a match.
4 Put the transfer loop into the fire until it turns to a glowing orange to
sterilize.
5
6
7
8
9

Unscrew cap to the test tube with Escherichia Coli.

Rub the transfer loop on the slanted agar.
Measure 1 milliliter of water with a graduated cylinder.
Pour into a sterilized test tube.
Put transfer loop in water and spin in circles get the Escherichia Coli off of
the loop and into the water.
10 Write Starter on the bottom of a petri dish.
11 Pour water onto agar inside of the petri dish.
12 Spin the petri dish in small circles so the water covers the entire
bottom of the petri dish.
13 Close the petri dish.
14 Turn the petri dish upside down and place in a 37 incubator.

Deluding the Ethyl Alcohol:

1 Pour 10 ml of 70% ethyl alcohol into a beaker.
2 Add 1 ml of water to the beaker to make the mixture about 69% ethyl
alcohol.
3 Repeat steps on and two, but instead of 1 ml, put in 3 ml of distilled water
to make it around 68% ethyl alcohol.
Procedure:
1 Soak a seven mm paper disk in the 68, 69, and 70% ethyl alcohol for one
minute.
2 Place three soaked paper disks in a petri dish that already has steps 1-11
in the bacteria prep section in it.
3 Label it on the bottom based on the temperature of the incubator it is
going in and the percentage of ethyl alcohol on the paper disks.
4 Place in its designated incubator and wait 24 hours.
5 After 24 hours take out the dish and count the number of Escherichia Coli
colonies around each disk.
6 Record the data in a chart.

Data and Observations

Table 1
DOE
DOE
1
2
3
Average

(+,+)
8 2/3
9
10
9.22

(Ethyl Alcohol, Temperature)

Standard
(+,-)
(-,+)
(-,-)
8 2/3 16 1/3 15 1/3
10 1/9
8 18 2/3 13 2/3
11 2/9
9 18 1/3 14 1/3
11
8.22
17.11
14.11
10 7/9

Table 1 above shows the averages of all three DOE tables. The first DOE
was run first and so on. To find all the numbers, the numbers from each run were
added together and then divided by the number of runs (3). All the numbers from
the DOE were consistent.

Table 2
Factors
(+)
70%

Standar
d
69%

(-)
68%

(+)
38C

Standar
d
37C

(-)
36C

Table 2 above shows the factors that were tested in the DOE experiment.
The Ethyl alcohol values were chose because Ethyl Alcohol is sold at 69% and
70% is the highest amount that can be bought and 68% is in some brands of
hand sanitizer. The temperature values were chosen because 37C is the

average temperature of the human body and the other two temperatures are an
even amount in difference from it. This was also referred to in the problem
statement.

Table 3.
DOE 1
Order
1
6
5
4
2
3
7

Run 1
Trial
Stand
(+,+)
(-,-)
Stand
(+,-)
(-,+)
Stand

Order
1
3
5
4
6
2
7

Run 2
Trial
Stand
(+,+)
(-,-)
Stand
(+,-)
(-,+)
Stand

Result
10
8
16
10
9
14
11

Table 4.
DOE 2
Order
1
6
5
4
3
2
7

Trial
Stand
(+,+)
(-,-)
Stand
(+,-)
(-,+)
Stand

Run 1
Trial
Stand
(+,+)
(-,-)
Stand
(+,-)

Order
1
6
2
4
6
5
7

Run 3
Trial
Stand
(+,+)
(-,-)
Stand
(+,-)
(-,+)
Stand

Result
11
8
15
13
8
17
11

Average
11
8 2/3
15 1/3
11
8 2/3
16 1/3
10 2/3

Result
12
10
15
10
9
18
10

Result
9
10
14
12
9
15
10

Order
1
6
3
4
2
5
7

Trial
Stand
(+,+)
(-,-)
Stand
(+,-)
(-,+)
Stand

Result
12
8
12
11
8
20
12

Order
1
2
3
4
6
5
7

Trial
Trial
(+,+)
(-,-)
Stand
(+,-)
(-,+)
Stand

Result
11
9
15
11
7
17
12

10 2/3
9
13 2/3
11 1/3
8
8 2/3
11 2/3

Result
10
9
15
11
11

Order
1
2
5
4
3

Run 2
Trial
Stand
(+,+)
(-,-)
Stand
(+,-)

Result
13
9
12
10
8

Order
1
6
3
4
5

Run 3
Trial
Stand
(+,+)
(-,-)
Stand
(+,-)

Result
10
9
16
12
8

Average
11
10
14 1/3
11
9

Table 5.
DOE 3
Order
1
6
5
4
3

2
7

(-,+)
Stand

16
10

6
7

(-,+)
Stand

20
11

2
7

(-,+)
Stand

19
12

Table 3. 4. And 5. Above show the results that were achieved when all of
the DOE trials were completed. When looking at the data there was on obvious
decrease in the amount of colonies when the Ethyl Alcohol was held at 70% or
the high amount.
Table 6
Observations
Day
Observations
The Escherichia coli is gathering around the paper tabs
1
like expected. The Escherichia coli has a really bad smell
to it.
The experiment is going as expected. There seems to be
more Escherichia coli around the low amount of Ethyl
2
Alcohol than the high amount. The Escherichia coli has a
really bad smell to it.
The standards are all in a small range, proving that the
3
experiment is valid. The Escherichia coli has a really bad
smell to it.
Table 6 above shows the observations that were recorded during the
experiment. The experiment only took three days to complete because three
runs were run each day. During the experiment, it was present that the high
amount of Ethyl Alcohol was allowing less Escherichia coli to grow around the
paper tabs.

18 1/3
11

(+,
+)
(S,S
)
(-,-)

Figure 1. Petri Dish Samples

Figure 1 above shows samples from DOE 2. The concentration and the
density of the bacteria is different in every dish. The bottom disk of the top three
has the most bacteria around all of the paper tabs because this petri dish was the
(-,-). The top right disk, (+,+), and the top left disk, (S,S), are about the same, but
the top right has a little bit less bacteria on it. All of the petri dishes had about the
same amount of coverage and a very similar number of colonies on it.
To compare the amount of colonies on each of the plates, colonies were
counted. Each plate that had the same treatment on it was very close in the
number of colonies that were able to grow on it.

Data Analysis and Interpretation

Data Analysis
Table 7
Factors
Ethyl Alcohol %
(+)
Standard
(-)
70%
69%
68%

Temperature C
(+)
Standard
(-)
38C
37C
36C

Table 7 above shows the values for the standard, high, and low for the
experiment. The high for Ethyl Alcohol was 70%. The standard was 69%, and
the low was a 68%. The high for Temperature was 38C, the standard was 37C,
and the low was 36C. The standards were chosen because they were body
temperature, and the percent used in many hand sanitizer products.

Number of Colonies
Standards

0 3 6 9 12 15 18 21 24 27 30
Standard Trial

Figure 2. Standards Scatter Plot

Figure 2 above shows the range of standards. The range of standards is
two. The highest standard was 12 and the lowest was 10. This shows that the
standards were consistent.
Table 8.
DOE Summary

Runs
Ethyl
Alcoho
l
(+)
(-)
(+)
(-)

Temperatur
e
(+)
(-)
(-)
(+)

First
DOE

8 2/3
15 1/3
8 2/3
16 1/3

Second DOE

Third DOE

9
10
13 2/3
14 1/3
8
9
18 2/3
18 1/3
Grand Average

Average

9.22
14.11
8.22
17.11
12.165

Table 8 above shows the average number of colonies for each of the
DOEs. The average number of colonies that grew in the high, high run was
9.22. The average number of colonies in the low, low run was 14.11. This shows
the difference in the amount of colonies that were able to grow in the conditions
of the Ethyl Alcohol and the temperature.

Table 9
Effect of Ethyl Alcohol

Ethyl Alcohol %
(-)

(+)

17.11

9.22

14.11
8.22
Average = 15.61
Average = 8.72
Effect= -6.89
Table 9 above shows the effect of ethyl alcohol. With the low amount
there was an average of 15.61 colonies. With the high amount, there was an
average of 8.72 colonies. With this data, there was an effect of -6.89, proving
that the data was significant, because two times the range of standards is 4 and
the absolute value of the effect of ethyl alcohol is bigger than two times the range
of standards. This proves that as Ethyl alcohol increased, the number of
colonies decreased.

Number of Colonies

-2

18
16
15.61
14
12
10
8
6
4
2
0
-1
0

8.72
Ethyl Alcohol

Effect of Ethyl Alcohol

Figure 3. Effect of Ethyl Alcohol

Figure 3 above also shows the effect of Ethyl Alcohol. This gives more of
a visual on the effect showing how as Ethyl Alcohol concentration increased, the
number of colonies that were able to grow decreased.

Table 10
Effect of Temperature
Temperature C
(-)
(+)
14.11

Table 10 above shows the effect of

9.22
temperature. When the amount was low,

8.22
17.11
Avg =11.17
Avg = 13.17
Effect = 2

the average number of colonies was

11.17. When the amount was held high,

the average number of colonies was 13.17 making the effect of temperature 2
colonies.
14

13.17

12
11.1710
8
Number of Colonies

Temperature

4
2
-2

-1

0
0

Effect of Temperature

Figure 4 Effect of Temperature

Shown above is the effect of temperature. To find the effect of temperature
you take the high minus the low. The effect is 2. This shows that as temperature
increases, the number of colonies able to grow also increases.

Table 11
Interaction Effect
Temperature C
(-)

(+)

%Ethyl Alcohol

Line Segment Solid

(+
)

8.22

9.22

Line Segment Dotted

(-)

14.11

17.11

Table 11 above shows the interaction effect of temperature and Ethyl

Alcohol. The table shows that when both of the low amounts of both factors were
used, there was an average of 14.11 colonies. When the amounts were both
high, there was an average of 9.22 colonies showing a decrease in the number
of colonies.
18
16
14
12
10
# Of Colonies

Ethyl Alcohol +

Ethyl Alcohol -

4
2
0
-1

1
Temperature

Figure 5 Interaction Effect

The above figure shows the interaction effect between ethyl alcohol and
temperature. The dotted line represents the low ethyl alcohol concentrate and the
solid line represents the high ethyl alcohol concentrate. The start of the line, on
the left, is where temperature is low and the end of the line, to the right,

represents where temperature was high. To find the interaction effect you take
the slope of the high line minus the slope of the low line. The interaction effect in
our case is -1.

-6

-4

-2

Figure 6 Dot Plot of Significance

Shown above is the dot plot of significance. Each arrow represents an
effect. E being ethyl alcohol, T is temperature, and ET is interaction. The lines
represent two times the range of standards. Anything outside of the lines is
significant. So it can be seen that only Ethyl alcohol was mathematically
significant.
Interpretation
The first factor in the experiment was Ethyl Alcohol. The Ethyl Alcohol was
tested at 68%, 69%, and 70%. Also, when the factor was held high, the average
number of colonies was 8.72 colonies. When the factor amount was low, there
was an average of 15.61 colonies. This gives Ethyl Alcohol an Effect of -6.89.
This can be referred to in Table 9. This shows that because the effect was

negative, when the percent of Ethyl Alcohol went up, the amount of colonies went
down, and therefore, Ethyl Alcohol does kill bacteria, even if the difference in the
different in concentration percent is as small as 1%.
The second factor in the experiment was temperature. The different
amounts that were used were 36C, 37C, and 38C. (When the amount was
held low, there was an average of 11.17 colonies. This can be referred to in Table
3.) When the amount was held high, the average number of colonies that grew
was 13.17. This can also be referred to in Table 10. This gives temperature an
effect of 2. This means that when temperature rises, Escherichia-coli is able to
grow faster and produce more colonies.
The last graph is the interaction effect. This shows how the effect of Ethyl
Alcohol and how the effect of temperature worked together. The comparison of
the high effect value of ethyl alcohol and the high effect value of ethyl alcohol
with temperature involved, the number with high temperature goes up slightly,
and the number with low temperature went down slightly. So while the interaction
effect is not mathematically significant, it does play a small role.
Lastly, is the importance of the standards scatter plot. The importance of
the Standards Scatter Plot is to see if the standards are all similar. For example
the graph should not look like it is snowing, so the standards are all relatively
close to each other. If the standards in an experiment are all far away from each
other, it means there was a problem in the experimental design or there was an
error made. If you refer to Figure 2, the standards were all close and it looks like
a relatively straight line.

Conclusion

The hypothesis that if colonies of E-Coli are exposed to 68% ethanol and
are placed in a 38C incubator, then the most colonies of Escherichia-coli will
grow was accepted.
This experiment was designed to test the effectively of Ethyl Alcohol
based hand sanitizers. This experiment tested the amount of bacteria colonies
that were able to grow in three different environments, all with a different alcohol
percentages. It was expected that when the Ethyl Alcohol value was low, the
amount of colonies would be a lot higher than when the Ethyl Alcohol value was
high. The environments varied with three different temperatures being 36C,
37C and 38C. When all three trials were averaged together, the high amount of
Ethyl alcohol and the high temperature allowed the second least colonies to grow
being 9.22. Ethyl alcohol high and temperature low allowed the least amount of
colonies to grow being 8.22. When Ethyl alcohol was held low and temperature
was high, 17.11 colonies were able to grow being the most amounts of colonies.
When Ethyl alcohol was low and temperature was also low, the second most
colonies were able to grow being 14.11. With the results that were achieved, it
was shown that the lowest percent of Ethyl Alcohol and the highest temperature
would allow the most colonies to grow.
This data showed that when the environment contained the least amount
of Ethyl alcohol, the most amount of colonies were able to grow. Also, when
temperature was higher, the colonies were able to reproduce faster. This is
logical because hand sanitizers with Ethyl Alcohol as the only active ingredient

were designed to kill all of the bacteria it comes in contact with and was
advertised to kill 99.9% of germs.
Throughout this experiment, there were a few design flaws. One of the
problems was that the distillation of Ethyl Alcohol was no exact. Another
example could have been that the amount of time each petri dish was in the
incubator varied by a few hours each day. This was caused by the scheduling of
the time of the experiment was conducted and the amount of time it took to reset
each of the trials. The last design flaw was that the variables were simply too
close together. Instead of using 68%, 69% and 70% Ethyl Alcohol, the
experiment could have been conducted with 50%, 60%, and 70% Ethyl Alcohol.
If the amounts were spread more, the results could have been more clear in
distinct to show that the variables were significant.
During the experiment, there were a few errors that were made. One error
was that the petri dishes were not completely sterile. Another example was that
the bottles the solutions were kept in were not completely sterile making the
solutions become contaminated. To avoid this, the solution bottles could have
been boiled and the solution could have been tested to see if they were
completely sterile. Also, the temperature of the incubators varied by about half a
degree throughout the trials..
For research to be continued, other chemicals for dilutions could be
experimented with to see what is most effective. One idea is that the Ethyl
Alcohol could be mixed with Isopropyl Alcohol, which is another chemical used in
different types of hand sanitizer, to see what combination is most effective and

what costs the least to make. Other thing that could be tested include the
amount of time Ethyl Alcohol is effective before it evaporates and more bacteria
is able to grow where the alcohol once was. This experiment helps to prove that
the higher the amount of Ethyl Alcohol, the lower the amount of colonies is able
to grow. This can help the understanding of hand sanitizer or disinfectant wipes
and sprays.
The results achieved could help disinfectant companies or hand sanitizer
companies to ensure that their product is as effective as possible. Continuing
this experiment could help to find the lowest cost of antibacterial chemicals that
work the best. This can help to prevent more sickness or disease from spreading
in the community.

Work Cited
Chu, P.M.. "Ethanol." Webbook. NIST, 4 2 2013. Web. 4 Feb 2013.
<http://webbook.nist.gov/cgi/cbook.cgi?ID=64-17-5&Type=IRSPEC&Index=QUANT-IR,8>.
"Overcrowded Schools Require Extra Sanitation Efforts."School Talk.
School Sanitation, 4 Feb 2013. Web. 4 Feb 2013.
<http://www.schoolsanitation.org/overcrowded-schools-require-extra-sanitationefforts/10/>.
Huynh-Delerme, Cline, Catherine Artigou, Laurent Bodin, Robert Tardif,
Ginette Charest-Tardif, Ccile Verdier, Nessryne Sater, Mostafa Ould-Elhkim, and
Catherine Desmares. "Abstract." National Center for Biotechnology Information.
U.S. National Library of Medicine, 17 Apr. 2012. Web. 05 Feb. 2013.

Acknowledgements
Mr. Estapa
Thank you for helping the experimenters to fix the design errors and for
the idea to count the colonies instead of resistance.
Mrs. Hilliard
Thank you for helping the experimenters format the paper and to complete
the experimental design.
Mrs. Dewey
Thank you for helping the experimenter to put the finishing touches on the
paper and to correct all of the formatting.