The Effectiveness of Mouthwashes Against the ... - Franklin College

The Effectiveness of Mouthwashes Against the Bacteria
Bacillus subtilis & Staphylococcus epidermidis
Lauren Query
April 23, 2002
Abstract
In this experiment, several commercial mouthwashes were tested in order to find
the most effective against the bacterium Bacillus subtilis and Staphylococcus
epidermidis. The five mouthwashes analyzed were Scope Clean Mint Baking Soda,
Listerine, Cepacol, Rembrandt and Therasol with water as the control. The results shown
that Therasol was the most successful in killing the bacteria Bacillus subtilis and Scope
and Therasol tied for the most effective against Staphylococcus epidermidis, while
Cepacol was very close (refer to Table 3). When Scope, Cepacol and Therasol were
analyzed, it was found that saccharin was a common component of the Cepacol and
Therasol and that polysorbate 80 was common in Therasol and Scope. All three
mouthwashes contained alcohol at differing percentages.
Introduction
A common problem that many people face everyday is that of Halitosis, or bad
breath. Many people attempt to conquer this situation by eating mints, chewing gum or
using mouthwash. One way bad breath occurs is by the decomposition of bacteria in our
mouth and throat regions. When the compounds in our mouth begin to breakdown, they

elease sulfides, which are produced by either grain negative bacteria or the renewal of
the oral mucosa tissue. A bi-product of the bacteria is hydrogen sulfide gases, which
produce volatile sulfide compounds (VSC) (Halitosis).
The question I asked myself was, “Which mouthwash will kill the most bacteria?”
My hypothesis for this experiment was that Listerine would inhibit the growth of the
most bacteria for the reason that when people have their tongues pierced, they are told to
use Listerine and it contains the highest percentage of alcohol.
The bacteria Bacillus subtilis is commonly found in the soil (Foundations in
Microbiology 273). If persons with a weakened immunity obtain Bacillus subtilis they
can get lung and blood infections (Microbiology 507). Bacillus subtilis produces spores,
which are dormant structures that serve as a way of reproduction. It is used as a
microbial source for the pharmaceutical Bacitracin, which is used as an antibiotic
effective against gram-positive bacteria (Foundations in Microbiology 748).
The well-known bacteria Staphylococcus epidermidis is most commonly found in
the skin, hair follicles and the mucous membranes (Foundations in Microbiology 482). It
is located along the nasal passages that are included in the normal flora. The normal flora
consists of the microorganisms that develop in the interior and exterior portions of the
body without producing obvious harmful effects on the tissues (Microbiology 335). The
genera Staphylococcus principally consists of gram-positive cocci and is normally present
on the skin (Microbiology 424).
2

Materials & Methods
The method of this experiment was described in the “Microbiology Lab:
Comparison of the Antibacterial Activity of Several Mouthwashes” by Dr. Steve
Browder, where the bacterium Bacillus subtilis and Staphylococcus epidermidis were
analyzed. An additional handout was given, listing the different types of mouthwashes to
analyze and the components found in them. The only test that was not performed was the
saliva test, which the information given was not acknowledged. The sterile techniques
that needed to be followed were not included in the lab handout. In order to ensure
sterility, the pipets were only touched at the end that does not come in contact with the
bacteria. The forceps that were used to transfer the filter paper disks of mouthwash were
first squirted with alcohol to kill any contamination. Also, keep the petri dish covered as
much as possible so that no other bacteria may contaminate the petri dish. After mixing
the bacteria and the agar in a test tube, the top of the tube was run through the open flame
of a bunson burner to kill any unwanted bacteria.
Results
The results found from this experiment are that Therasol was the most effective
against Bacillus subtilis and inhibited the growth by an average of 16.5 mm (refer to
Table 3). The data resulted in Therasol as the number one mouthwash, Scope was
number two, and Cepacol and Rembrandt are tied for number three, and Listerine was
number four for Bacillus subtilis.
3

Scope and Therasol tied for number one, Cepacol was number two, Rembrandt
was number three, and Listerine was number four for Staphylococcus epidermidis. Scope
had the largest inhibition ring of 19.5 mm and Therasol and Cepacol were close with 16.5
mm and 13.9 mm, respectively (refer to Table 3).
Table 1 lists the zone of inhibition measured in millimeters for all the
mouthwashes performed by all the groups for Bacillus subtilis. Table 2 shows the
information for the zone of inhibition of Staphylococcus epidermidis of the mouthwashes
for all the groups. Table 3 represents the average growth of inhibition for each type of
mouthwash for both of the bacteria. To measure the growth of inhibition, the distance
from the end of the mouthwash disk to the widest point of inhibition was measured using
a ruler in millimeters (mm). It also states the standard deviation for each mouthwash for
both of the bacteria. Table 4 lists the tabulated T-values for each mouthwash compared
to one another for both of the bacteria. It also shows the degrees of freedom at the 95%,
99% and 99.9% confidence levels.
Graph 1 illustrates the mean for zone of inhibition for each type of mouthwash for
Bacillus subtilis. Graph 2 represents the mean for zone of inhibition for they different
types of mouthwashes for Staphylococcus epidermidis.
Discussion
After the data was analyzed, the results did not represent the initial hypothesis.
The hypothesis stated that Listerine would be the most effective at inhibiting the growth
of the bacteria. Listerine was proven to be the least effective mouthwash by inhibiting
zero growth of the bacteria, which was the same for water (refer to Table 3). Since
4

Listerine contained the most alcohol (26.9%) it shows that alcohol was not as effective as
the other components.
When the mouthwashes were analyzed more closely, I found similar components
that might factor into the effectiveness they had on the inhibition of bacteria growth.
Scope and Cepacol both contain polysorbate 80 and cetyl pyridinium chloride.
Polysorbate 80 is used as a pharmaceutical aid, emulsifier and dispensing agent in
medical products and a defoamer and emulsifier in foods (Budavari 1207). This evidence
gives reason to believe that polysorbate 80 is not an antibacterial agent. Cetyl pyridinium
chloride is used as a topical antiseptic and as a disinfectant, which gives is it germ killing
power (Budavari 311).
The two common components found in Therasol and Cepacol was saccharin and
glycerin, while Scope contained sodium saccharin. Saccharin acts as a sweetening agent
in many food products, which is not antibacterial (Budavari 1321). Glycerin is produced
from sugars by fermentation and is used in liquid soaps, which can have an antibacterial
effect (Budavari 705). Listerine does not contain polysorbate 80, saccharin or glycerin,
which the leading mouthwashes contained. All three mouthwashes contain different
amounts of alcohol, which is used as an antibacterial agent. It can be used to cleanse
various equipment and wounds. While alcohol is a good bacteria killing agent, it has a
drying effect of oral tissue and makes the mouthwash effects short lived. When the
mouth becomes dry, it aids to the growth of bacteria, which leads to worse bad breath
than before (Halitosis).
After analyzing the components of Therasol, I found that sodium fluoride is used
as in pesticide formulations and as an insecticide to kill ants and cockroaches (Budavari
5

1361-1362). This might explain why Therasol worked best when used against Bacillus
subtilis. Since the bacterium is commonly found in the soil and pesticides are used to kill
insects the pesticides will eventually reach the soil through rain, killing the bacteria.
Neither Scope nor Cepacol contain this ingredient.
When Bacillus subtilis was analyzed using statistical analysis, it was found that
Therasol and Scope had a 95% significant difference level. Therasol and Cepacol had a
99.9% significant difference level. Scope and Cepacol had a significant difference level
of 99%. Rembrandt and Cepacol had no significant difference, meaning they are tied at
the third best mouthwash. This data concludes that Therasol is the number one
mouthwash, Scope was number two, and Cepacol and Rembrandt are tied for number
three, and Listerine was number four.
When the data was statistically analyzed for Staphylococcus epidermidis, the data
shown that Scope and Therasol showed no significant difference between them even
though Scope’s mean was higher than that of Therasol. This means that Scope and
Therasol tied for the significance of killing data. When Therasol and Cepacol were
analyzed through statistical analysis, it was found that there is no significant difference
between the two mouthwashes. When Scope and Cepacol were analyzed, it was found
that there was a 95% significant difference. For Cepacol and Rembrandt there was a 95%
significant difference. This data shows that Scope and Therasol tied for number one,
Cepacol was number two, Rembrandt was number three, and Listerine was number four
for Staphylococcus epidermidis.
Since glycerin is an antibacterial disinfectant in liquid soaps, this gives reason as
to the ability of Cepacol and Therasol to inhibit the growth of the bacteria Staphylococcus
6

epidermidis. Even though Scope does not contain glycerin and was the most effective
against Staphylococcus epidermidis, the Scope contained cetyl pyridinium chloride,
which gives it the ability to kill bacteria. Cepacol and Therasol also contained saccharin,
which does not give it any killing power because it acts as a sweetening agent.
After researching the mouthwashes and bacteria, it makes sense that Listerine was
not an effective mouthwash because the bacteria examined are not located in the mouth
or throat regions. In order to have an accurate experiment examining mouthwash, the
bacteria living in the mouth and throat regions should be the ones tested.
7

Works Cited
“Bad Breath” N.D.
Browder, Steve. Microbiology Lab: Comparison of the Antibacterial Activity of Several
Mouthwashes. Mar. 2002.
Budavari, Susan, et al. The Merck Index: An Encyclopedia of Chemicals, Drugs, and
Biologicals. 11th ed. New Jersey: Merck and Co., Inc., 1989.
“Halitosis” Herskovits, Gary. 18 Apr.
2002.
Nester-Roberts-Nester. Microbiology. Iowa: Wm. C. Brown Communications, Inc.,
1995. pp. 335 and 424.
Talaro-Talaro. Foundations in Microbiology. Iowa: Wm. C. Brown Communications,
Inc., 1993. pp. 79, 273, 487, 501 and 748.
8

Mean Zone of
Inhibition (mm)
Mean Zone of
Inhibition (mm)
18
16
14
12
10
8
6
4
2
0
25
20
15
10
5
0
Bacillus subtilis
Mean Zones of Inhibition
water
Scope
Listerine
Cepacol
Rembrandt
Therasol
Type of Mouthwash
Staphylococcus epidermidis
Mean Zones of Inhibition
water
Scope
Listerine
Cepacol
Rembrandt
Therasol
Type of Mouthwash
Graph 1
Series1
Series1