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Research Question: How does the concentration of ethanol in Listerine mouthwash (0%, 10%,
20%, 30%, 40%, 50%) affect the inhibition of the growth of Escherichia coli after 48 hours through
measuring the zone of inhibition of the E.coli surrounding a mouthwash infused paper disk?

1: Introduction
Bacteria are essential organisms within the ecosystem that can be found both inside and outside of the
human body, however, whilst some bacterial strains have positive effects on the human body, others
have negative effects. For this reason, we must ensure that we keep our bodies clean to avoid the
growth of such bacteria. The system in our body that is most responsible for attracting bacteria in our
mouth. When bacteria enter our mouth it feeds on food remnants between our teeth and on our tongue,
this can cause tooth decay or oral infections and may progress to further illnesses depending on the
bacterial strains (Oral Hygiene, 2021). Therefore, it is crucial to clean your mouth daily with both
toothpaste and mouthwash. Brushing your teeth and flossing ensures that food remnants are removed,
whilst mouthwash is responsible for directly killing bacteria. Mouthwash consists of several
ingredients aimed to kill bacteria, some of these include fluorine and chloride, additionally, some
mouthwash brands choose to include ethanol whilst other brands choose not to.

1.1: Personal Engagement

I started to become more aware of my hygiene due to the COVID-19 pandemic, where I was
constantly told the importance of hand sanitizer and keeping your hands away from your mouth. I
became increasingly cautious of not only the COVID-19 virus but also other viruses and bacteria that
may harm my body. Several times a day I began rubbing sanitizer onto my hands, yet I noticed that
my oral hygiene remained the same. I wanted to know which additional preventative measures I
should be taking in order to keep harmful bacteria and viruses away from my body and which day-to-
day products are most effective to do so. During my research, I noticed that only some mouthwash
products contain ethanol whereas it is the fundamental component of other products such as hand
sanitizer. I was curious about the reason why ethanol is only used in some mouthwash products and
wanted to understand whether it was more effective than non-alcoholic mouthwash or if the other
ingredients were just as effective and ethanol was unnecessary. I decided to conduct an experiment
with the common bacteria Escherichia coli using the Kirby-Bauer method. In order to conduct a fair
experiment, I used the same mouthwash solution and measured out and combined the different
ethanol concentrations myself so all other chemicals are standardized for each solution.

1.2: Preliminary experiment

Prior to the experiment, I conducted a preliminary experiment to assess whether any alterations were
necessary to the Kirby-Bauer method to ensure maximum efficiency and accuracy of results. In the
preliminary experiment, I placed all the paper disks in the different mouthwash solutions at the same
time with the intention of removing them at the same time as well. However, the process of removing
the paper disks with a tweezer came at a greater challenge than initially expected, which led to a
difference of time that each disk was soaking for. A difference in soaking time would have been an
extraneous variable, thus for the final experiment, I chose to only soak four disks at a time in the same
solution so that the difference would not exceed an insignificant time.

2: Investigation
2.1: Background Knowledge
Ethanol is often used as a universal disinfectant where it can be found within soap, mouthwash, and
hand sanitizer. It kills bacteria through the process of membrane denaturation (Flournoy, B. 2020).
Bacteria are surrounded by a membrane built up of a chain of fatty acids, this membrane is
hydrophobic, meaning that it will not dissolve in water, thus, the membrane stays intact in the
presence of water. However, ethanol (C2H5OH) is an organic solvent and an amphiphile chemical
compound, meaning it entails both hydrophilic and lipophilic properties. The polar end of ethanol
−¿¿
(O H ) dissolves well with water whilst the nonpolar end (C2H5) dissolves lipids (Ribeiro et al,

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2015). Meanwhile, bacterial structures consist of water on the inside and lipids on the membrane.
Therefore, when bacteria are in the presence of ethanol the fatty acid chain will begin to dissolve and
the bacterial membrane will be broken apart. Subsequently, the water within the bacterial cytoplasm
will dissolve in the ethanol as well. Thus, the bacteria will rupture, which means that the core contents
of the bacteria will be exposed, losing their structure and disabling their function, leaving them to die
(Ingram L. O. 1990). Because of this process of membrane denaturation, ethanol is globally trusted to
inhibit bacterial growth within both scientific laboratories and within household products such as
Listerine mouthwash. However, alcoholic Listerine mouthwash usually only contains about 26%
ethanol, other ingredients include water, benzoic acid, menthol, methyl salicylate, sodium fluoride,
and others. These ingredients function to strengthen your teeth, clear your breath and along with
ethanol, kill off bacteria and other pathogens (Listerine, 2020).

2.2: Hypothesis
H1: As the concentration of ethanol increases, the zone of inhibition (mm) of E.coli around the
mouthwash-infused paper disks will increase after 48 hours of growth.

Fig 1. The directional hypothesis with rough values

Justification of Directional Hypothesis:

Ethanol is proven to kill most bacteria, so an increase in ethanol concentration in Listerine mouthwash
will increase the bacterial inhibition of E.coli.

H0: There is no correlation between the concentration of ethanol in mouthwash and the zone of
inhibition (mm) of E.coli around the mouthwash-infused paper disks after 48 hours of growth.

2.3: Variables
2.3.1: Independent Variable
The ethanol concentration of Listerine mouthwash.
Units: Percentage of ethanol : 0%, 10%, 20%, 30%, 40%, 50%
Justification: Store-bought mouthwash ranges from 0%- 40% ethanol, so ethanol concentrations
accurately reflect real mouthwash ingredients. Percentages were chosen in exact increments of 10 in
order to produce an appropriate graph.

2.3.2: Dependent Variable

The zone of inhibition of the E.coli in millimeters around the mouthwash-infused paper disks in the
agar plate.
Units: Millimeters
Further calculations: Pearson's correlation coefficient and Spearman's rank correlation coefficient.

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2.3.3: Controlled Variables

Controlled Variable Significance Method of Control

The time the paper May alter the amount of mouthwash Used a stopwatch to accurately
disks soak in infused in the paper disk which may alter measure the amount of time
mouthwash the bacterial inhibition soaking in mouthwash

Source of E.coli Mutations may arise in different E.coli Used the same E.coli culture for
solutions which may give some bacteria a each agar plate
stronger resistance towards ethanol than
others

The volume of A larger volume of E.coli in an agar plate Used a micropipette to accurately
E.coli spread over would have increased the bacterial growth measure out 50μm of E.coli for
each agar plate each agar plate

The temperature of Temperature has a direct effect on Placed all agar plates in the same
the surrounding bacterial growth, different temp would 28°C incubator
environment have altered the rate of bacterial growth
for different agar plates

Time of bacterial Agar plates with more time to grow
growth would have a larger E.coli culture than
those left for a shorter time
Ensured that all agar plates were
prepared with E.coli
simultaneously and zone of
inhibition was measured in the
same sitting

Exposure to external External bacteria may have a different All apparatus was sterilized
bacteria resistance towards the ethanol and alter before coming in contact with the
the zone of inhibition E.coli or agar plate. The Agar
plate was only opened at a 45°
angle to avoid exposure

Type of mouthwash Different mouthwash brands have Used a standardized Listerine

different ingredients, therefore, some may non-alcoholic mouthwash for all
be more effective than others and so the solutions
ethanol would not be the only independent
variable

3: Procedure
3.2: Materials and Apparatus
1. 250ml Ethanol
2. 450ml Non-alcoholic Listerine mouthwash
3. 1 Bunsen burner
4. 1 Heat mat
5. 8 Agar plates
6. 50ml E.coli solution
7. 1 Micropipette
8. 8 Micropipette tips ±2.0 μL
9. 7 100ml Beakers
10. 2 100ml Measuring cylinders ±0.1ml
11. 8 Plastic Spreaders
12. 32 Paper disks

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13. 1 Tweezer
14. 1 28°C incubator
15. 1 Ruler ±0.5mm
16. 1 Permanent marker
17. 1 Stopwatch

3.3: Method
Prepare the apparatus:
1. Sterilize the worktop with ethanol
2. Fill a 100ml beaker with 50ml ethanol
3. Set up a bunsen burner on a heat mat on low intensity
4. Split agar plate into four equal sections by marker lines on the bottom of the agar plate
5. Label the ethanol concentration on each section (5 sections for each concentration) with a
permanent marker

Prepare the E.coli:

6. Shake the E. Coli solution for 30 seconds
7. Adjust micropipette to 50μm and prep micropipette into a sterile tip and flame top of E. coli
bottle for sterilization
8. Press stopper at the top of the micropipette to the first point and hold, then place in E.coli
solution to draw it up into the micropipette
9. Open agar plate at a 45° angle and release the stopper to eject E.coli into an agar plate
10. Eject the tip into a waste beaker by pressing on the stopper twice
11. Use a new and sterilized plastic spreader to spread E.coli evenly onto the agar plate, keep the
lid at a 45° angle
12. Repeat steps 6-11 for six separate agar plates using the same E.coli solution

Measure out the solution:

13. Prepare six 100ml beakers
14. Use a measuring cylinder to mix the mouthwash and ethanol following the table below :
0% 10% 20% 30% 40% 50%
ethanol ethanol ethanol ethanol ethanol ethanol

Ethanol 0ml 5ml 10ml 15ml 20ml 25ml

Mouthwash 50ml 45ml 40ml 35ml 30ml 25ml

15. Sterilize tweezers in ethanol and hold them over the low-intensity flame for 5 seconds, (let it
cool off before usage)
16. Place 4 individual filter paper disks into each solution with the tweezers and let it soak for 1
min (Do this one solution at a time), use a stopwatch to record the time
17. As there is only space for four disks per agar plate, soak one extra paper disk in each solution
at the end to place in the same agar plate
18. Take the filter paper disks out and let them dry on a sterilized surface for 30 seconds
19. Lightly place one filter paper disk on each section on the agar plate with sterilized tweezers as
seen below (keep agar plate lid on a 45° angle)

Fig 2. Set up of agar plates

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20. Tape lid onto agar plates

21. Place all agar plates in a 28° C incubator facing upwards and leave for 48 hours

Measure results:
22. After 48 hours, take out all agar plates from the incubator
23. Use a ruler to measure the inhibition zone around the paper disk from one end to another in
mm through the agar plate lid
24. Write down each value in a table

Disposal:
25. After usage, throw all disposable materials including agar plates into a disposable bag
26. Sterilize the worktop with ethanol
*Adapted from Kirby-Baurer method

3.4: Set up
Fig 3. Photograph of the final agar plate with 20% ethanol concentration mouthwash disks

3.5: Justification of Method

The Kirby-Bauer method was used and adapted as it allowed me to measure out and compare the
inhibition zones from the different mouthwash solutions in a standardized test. The mouthwash and
ethanol solutions were calculated and combined by hand to ensure that the percentages were exact in
intervals of 10%. Six ethanol percentages of 0%, 10%, 20%, 30%, 40%, and 50% were used as store-
bought mouthwash contains anywhere from 0% - 40% ethanol, thus, the solutions in the experiment
would accurately reflect real mouthwash ingredients. The same mouthwash was used for each
solution to ensure that the ethanol concentration was the only independent variable present in the
experiment rather than another chemical that would have varied in different mouthwash solutions.
The Kirby-Bauer experiment allowed numerical measurements to be determined by measuring the
zone of inhibition in millimeters.
5 repeats of each solution were carried out to improve the accuracy of the dependent variable data and
to ensure that the data collected did not include outliers. Escherichia Coli was used as it is a relatively
safe and harmless bacteria to the human body that grows quickly and is easy to culture, this ensured
that the experiment could be carried out in a couple of days. Additionally, E.coli is a common bacteria
often found in the mouth (Oliveira et al., 2012), making the results applicable to real-life scenarios.
The zone of inhibition was chosen as the dependent variable as it can be correlated with the resistance
of the solution towards the E.coli.

3.6: Risk Assessment

Safety Consideration Prevention

A bunsen burner flame was alight during the The flame was kept on a safety mat and away from
duration of the experiment for sterilization the immediate and close range of the apparatus and
which could have caused burns. experimenter.

The temperature of the incubator could burn The incubator temperature was set at 28° C to
the skin if accidentally touched, if too hot. avoid the risk of burns.

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Environmental Consideration Prevention

E.coli may be exposed to a water system and E.coli was handled away from all water sources,
create fecal pollution which can affect other and all tools which it came in contact with were
bacterial strains in the water as well as disposed of immediately after usage.
organisms that consume the water.

The plastic tools used get disposed of which All plastic tools were thrown in a disposal bag to
could contribute to plastic waste in the ocean get taken for combustion or landfill rather than into
or nature. the environment.

Ethical Consideration Prevention

The E.coli bacteria, though usually harmless, The E.coli was handled with disposable tools that
may be pathogenic. This means that they were placed in a waste bin immediately after usage.
may cause an illness if a person is exposed to The agar plate was additionally only opened at a
it. 45° angle to avoid exposure.

4: Results
4.1: Raw Data
Table 1. Raw data of zone of inhibition (mm) to the respective ethanol concentration of mouthwash.
Zone of inhibition (mm) ±0.5mm
Ethanol
concentration of
mouthwash (%) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
0.0 11.0 10.5 10.0 11.0 10.0
10.0 12.0 11.0 11.0 12.0 10.0
20.0 9.0 10.0 9.0 10.0 11.0

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30.0 9.0 8.0 9.0 9.0 9.0

40.0 12.0 12.0 11.0 12.0 10.0
50.0 9.0 10.0 9.0 9.0 8.0
*Each value rounded to nearest 0.5 millimeter

4.2: Qualitative data

1. The zone of inhibition of the E.coli surrounding the paper
disk formed an imperfect ring where some ends were
further away from the disk than others. This meant that
some measurements taken were inexact.
2. Zone of inhibition values differ within the same
concentration
3. The growth of the E.coli was uneven, where some areas of
the agar plate had a larger culture than others.

Fig 5. Agar plate after experiment

4.3: Raw Data Calculations

Mean

To find the mean values of the zone of inhibition (mm) of each ethanol concentration of mouthwash
I calculated,
All values of the zone of inhibiton(mm) of one concentration
= mean
number of values
Standard Deviation

To find the standard deviation of the values of the zone of inhibitions of each ethanol concentration
of mouthwash I calculated,
σ =√ ❑
σ =Standard Deviation
Σ=∑ of .. .
X =Each value
μ= Mean
N=Number of value∈ population

4.4: Processed Data

Zone of inhibition (mm)
Ethanol concentration of mouthwash (%) Mean (mm) ±0.5mm Standard deviation
0.0 10.5 0.50
10.0 11.2 0.84
20.0 9.8 0.84
30.0 8.8 0.45
40.0 11.4 0.89
50.0 9.0 0.71

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Table 2. Mean of the zone of inhibition to ethanol concentration and standard deviation of results.
*Mean rounded to the nearest tenth value - Standard deviation rounded to the nearest hundredth value

Fig 6. Scatter plot of processed data with the corresponding line of best fit and the R2value.

4.5: Statistical Test

The aim of the experiment was to investigate the relationship between the concentration of ethanol in
mouthwash and bacterial inhibition to determine whether the two variables have a correlation, thus,
using both a Spearman’s rank correlation coefficient test and a Pearson’s correlation coefficient test
was the most appropriate method to analyze the relationship between the two variables. Both
Pearson’s and Spearman’s tests give an r/rs value which uses the same scale that represents the
statistical significance and correlation direction. The r values range from -1 to +1:

Fig 7. Scale of r value

4.5.1: Spearman’s Rank Correlation Coefficient

Spearman’s test is represented as:

rs = Spearman’s rank correlation coefficient ( ρ )

D = Difference between the two ranks of observation
n = Number of observations

Spearman’s rank is a statistical method used to measure the strength of a correlational relationship
between two variables or sets of data by ranking the y-values and comparing the rank of the x-values,
rather than using the raw numerical values.
Calculation Using VassarStats:

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Fig 7. VassarStats Spearman’s Rank Calculations

4.5.2: Pearson’s Correlation Coefficient

Pearson’s correlation coefficient is represented as:

r = Correlation coefficient
x = Values of x
x̄ = Mean of the x-values
y = Values of y
ȳ = Mean of the y-values

Pearson’s correlation coefficient is a statistical method used to measure the strength of a correlational
relationship by evaluating a linear relationship between two variables.

Calculation Using VassarStats:

Fig 8. VassarStats Pearson’s Correlation Coefficient Calculations

4.5.3: Results of Statistical Tests

Using Spearman’s rank, it was determined that the rs-value was -0.257.
Using Pearson’s correlation coefficient, it was determined that the r-value was -0.383.
These values signified a weak negative correlation which was not statistically significant.

5: Data Analysis
The results from the experiment show that there is no significant relationship between ethanol
concentration in mouthwash and the inhibition (mm) of Escherichia coli growth. This was determined
as when the ethanol concentration increased from 0% - 50%, the zone of inhibition (mm) stayed
within 8.0 mm to 12.0 mm for each test without a significant directional increase or decrease.
Curiously, 30% ethanol concentration had a significantly smaller zone of inhibition than 40%,
however, it can be assumed that this is an anomaly caused by an extraneous variable such as the

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uneven growth of the E.coli. The standard deviation of the dependent variable values shows that the
results do not follow a significant pattern, where several error bars, which are based on their
corresponding standard deviation, overlap. This overlapping signifies that a certain measurement of
bacterial inhibition does not correlate with any specific ethanol concentration, yet may instead
correspond with multiple concentrations.
Furthermore, the processed data is unaffected by any uncertainties of the measuring equipment. The
uncertainties of the mouthwash solution make up 0.2% of the total solution, which would have an
insignificant effect. However, the uncertainty of the E.coli makes up 4% of the total volume and the
uncertainty of the zone of inhibition from the ruler is ±0.5mm, making up roughly 5%. These
uncertainties may have affected the raw data, yet due to multiple trials being completed, the effect
would be minuscule. Thus, the results of this experiment can be relied upon.

6: Conclusion
The results of this experiment disprove the directional hypothesis that the increase in ethanol
concentration will increase E.coli inhibition as there was no significant correlation between the
ethanol concentration in mouthwash and the zone of inhibition of E.coli. This can be supported by the
r values obtained from Pearson’s & Spearman’s statistical tests. With the r-value of -0.383 and the rs
value of -0.257, it can be established that there is a slight negative correlation, yet, it is not statistically
significant enough to determine a correlational relationship. Spearman’s rs value claims that there is a
weaker correlation than Pearson's r value as it focuses on the order of the y-values in relation to the x-
values, whereas Pearson’s test focuses on the linear regression which is based on numerical values.
Because of this, Pearson’s correlation coefficient is more constructed around individual values rather
than patterns and is more affected by outliers or anomalies. From the graph (Fig. 6), it can be
observed that there is no significant correlation between the dependent and independent variables as
no clear pattern or order can be detected. As Spearman’s rank is constructed around an order rather
than numerical values, it is the more suitable statistical measure to determine the statistical
significance of the results.
The results of this experiment occurred due to the other active chemicals in the Listerine mouthwash
such as sodium fluoride (Listerine, 2021) which are intended to kill bacteria as well. Sodium fluoride
is an antimicrobial that can inhibit bacterial metabolism through several mechanisms such as acting as
an enzyme inhibitor (Marquis, 1995). This inhibits the bacteria from obtaining energy which kills it.
Because of this, the effects of ethanol on bacteria, though effective on its own, becomes insignificant
in the presence of other antibacterial chemicals. The textbook theory would suggest that increasing
ethanol concentration would inhibit more bacterial growth as alcohol is effective on its own, however,
the data from this experiment showed that there is no correlation between the two. Thus, the zone of
inhibition (mm) around the mouthwash-infused paper disk did not increase or decrease after 48 hours
of bacterial growth in respect to the ethanol concentration.

A similar study was conducted at Tabriz University of Medical Sciences with the aim of
understanding the antibacterial properties of Chlorhexidine in mouthwash, yet they also tested the
difference between alcoholic and non-alcoholic mouthwash. This study found similar results to me
where there was no significant difference in bacterial inhibition between the alcoholic and non-
alcoholic mouthwash (Bahlouli et al., 2018). These were their results:

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Fig 9. Column Chart of Mouthwash Experiment

Other studies by dentists have suggested both that alcohol increases and decreases bacterial inhibition,
(Hale, 2021) yet the main argument against alcoholic mouthwash seems to be more psychological
than biological, where it is usually simply better in taste and feel (Exceptional Dentistry, 2020).

7: Evaluation
7.1: Strengths
Strength Effect on result
The initial mouthwash solution was No other chemicals altered the inhibition of bacterial
standardized growth

The ethanol concentration of the An appropriate graph was formed where logical
mouthwash was calculated by hand comparisons were able to be made between different sets
into exact increments of 10% of values

The common bacteria E.coli was used E.coli which would often be found in the mouth was used,
for the experiment because of this the results are applicable to real-life
scenarios

Real store-bought mouthwash was The conclusion can be applied to the usage of real
used mouthwash in real-life scenarios

7.2: Limitations
Limitation Effect on result Improvement
Measurements were Measurements may have been Take a photo of the agar plate
taken by a ruler and inaccurate as they were unable to be from a 90° angle with a vernier
eyeballed through the taken directly but through a transparent caliper in the photo to get an
agar plate cover. barrier and using an imperfect exact measurement.
measuring tool (ruler).

The zone of inhibition Measurements taken with a ruler would Measure the diameter using a
made an imperfect have been inexact and thus would have vernier caliper from two
ring. led to partially inaccurate data. different points to get an
accurate average of the zone of
inhibition.

The bacterial culture More difficult to establish the zone of Leave bacteria to grow for an

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was small and had not inhibition when the bacteria is not additional 48+ hours.
grown over the whole covering the whole surface.
agar plate.

E.coli was the only Different bacterial organisms may react Repeat the experiment using
bacteria used in the differently to ethanol. Thus, the results different common bacterial
experiment. are not fully applicable in a real-life strains found in the mouth such
context. as Streptococcus,
Granulicatella.

7.3: Extension
Mouthwash contains several different chemicals aimed to inhibit bacterial growth including ethanol.
Therefore, a valuable extension to further investigate which mouthwash ingredients are most effective
would be to repeat the experiment using other chemicals as the independent variable instead of
ethanol. Different mouthwash solutions containing different concentrations of fluoride or chlorine
could be tested and then compared. By investigating additional mouthwash chemicals, a more
informed conclusion could be made about which ingredients are the most effective in mouthwash.

Another possible extension would be to use different mouthwash brands which are premade as the
independent variable whilst using the same method. This allows a highly applicable conclusion to be
made, where the results would directly reflect actual mouthwash products and their individual
effectiveness.

6: Appendices
Appendix 1. Photo of results

0% Ethanol 10% 30% 50% 0%, 10%, 40%, 50%

20% 40%
Ethanol Ethanol Ethanol 20%, 30% Ethanol
Ethanol Ethanol
Ethanol

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