Saint Martin’s University
A Comparison of Over-the-Counter Mouthwashes and Their Effectiveness Against
Streptococcus mutans, A Common Oral Bacteria
Lisa Hall
Biology Senior Seminar
May 7, 2007
1
Abstract
This study was conducted to examine the antibacterial effectiveness of 10 over-thecounter mouthwashes on Streptococcus mutans, a common oral bacterium. The over-thecounter mouthwashes tested were Listerine®, Plax®, Walgreen’s Fresh Breath®,
Walgreen’s Fluoride®, Viadent®, Dr. Tichenor’s®, Scope®, Targon®, Breath Rx®, and
Crest Prohealth®. Four active ingredients were also tested at three different
concentrations per active ingredient, cetylpiridinium chloride (0.05%, 0.07%, 0.10%),
alcohol (10%, 20%, 30%), menthol (0.02%, 0.05%, 0.10%), and thymol (0.05%, 0.07%,
0.10%). The average zone of inhibition was measured in millimeters from the
mouthwashes and active ingredient concentrations; the data were collected from 20
replicates per treatment after an incubation period of 24 hours at 37°C. The results of a
one-way analysis of variance statistical test (ANOVA) showed a significant difference
among the mouthwashes (F = 34.63, df = 11, p-value 0.0005). A one-way ANOVA test
was performed on the data collected from the active ingredient dilutions and also found a
significant difference (F = 35.49, d.f. = 9, p-value = 0.0005). These results show that they
over-the-counter mouthwashes and active ingredients tested vary among their
antibacterial effectiveness. A Tukey multiple comparisons test was performed using
Minitab®. There were large variations in the sizes of zones of inhibition between the
mouthwashes, ranging from Walgreen’s Fresh Breath® at 0.0mm to Scope® at 14.6mm.
Scope® had the highest average zone of inhibition, and was significantly higher than
Targon®, Dr.Tichenor’s®, Breath Rx®, Plax®, Walgreen’s Fresh Breath® and Viadent®
with a 95% confidence interval. The cetylpyridinium chloride test groups showed a larger
zone of inhibition than all other active ingredients tested. This result supported the
findings that the cetylpyridinium chloride-containing mouthwashes tested also had higher
antibacterial effectiveness.
2
Table of Contents
I. Introduction
1-6
II. Methods
A. Agar Preparation for Petri Dishes
8
B. Growing Streptococcus mutans
8-9
C. Diluting Active Ingredients
9-11
D. Applying Mouthwash, Active Ingredient Dilutions,
or water control to Petri Dishes Containing Streptococcus mutans
12-13
E. Statistical Analysis
13-14
III. Results
14-16
IV. Discussion
16-19
V. Acknowledgments
20
VI. Literature Cited
21-22
3
I. Introduction
Many different oral bacteria contribute to diseases of the mouth. One of the most
common is Streptococcus mutans, gram positive, anaerobic bacteria that live in the oral
cavity (Scherp, 1971). S. mutans is a major contributor to dental plaque, because it is
able to adhere itself to the surface enamel of the teeth and makes up roughly 10 percent
of plaque’s dry weight (Scherp, 1971). Many people assume that brushing and flossing
their teeth is enough to keep them from developing diseases of the mouth, but an
antimicrobial mouthwash is needed to kill a greater number of harmful bacteria.
Antimicrobial mouthwashes are commonly used to reduce levels of oral pathogens and
there are many commercial mouthwashes, dental gels, and varnishes that are used to
combat oral bacteria (Menendez et al., 2005). Commercial mouthwashes contain many
active ingredients that claim to kill oral bacteria. For example, chlorohexidine is an
active ingredient that has become the gold standard for antimicrobial and anti-plaque
mouthwashes (Arweiler et al., 2006). It is often used as a positive control in studies of
oral bacteria, even though large variations between in vivo and in vitro studies have been
found in reducing levels of S. mutans (Menendez et al., 2005). Chlorohexidine is
available by prescription only in the United States, and many side effects accompany the
use of chlorohexidine solutions, such as burning of the mouth, drying of the oral mucosa,
possibility of carcinogenic effects, softening of some composite filling materials, and
staining of the teeth (Menendez et al., 2005). A 0.2% chlorohexidine solution has been
used successfully in Europe for more than 30 years; it has only recently been approved
for use in the United States and as a prescription in only a 0.12% solution (Menendez et
al., 2005). Since chlorohexidine’s introduction to the United States market, new
4
solutions have been introduced that promise better results with fewer side effects, but few
clinical trials have demonstrated the efficiency of these new solutions (Arweiler et al.,
2006). Over-the-counter mouthwashes are readily available and lack the side effects of
chlorohexidine products; the differences between these mouthwashes are their active
ingredients and the concentrations of these active ingredients.
Rinsing with an antimicrobial mouthwash and flossing are two hygienic
techniques that a small percentage of the American population performs (Zimmer et al.,
2006). According to a recent survey conducted by the American Dental Association
(2005), 42.2% of Americans say that they floss on a daily basis. Flossing is timeconsuming and painful to many people; the logical alternative is an antimicrobial
mouthwash to reduce plaque and the risk of gingivitis. Zimmer et al. (2006) compared
the anti-plaque effectiveness of flossing to that of antimicrobial mouthwashes. A total of
156 volunteers were randomly assigned to groups of brushing and flossing versus
brushing and rinsing with a mouthwash. After eight weeks, the volunteers were tested for
a reduction in oral bacterial growth (Zimmer et al., 2006). There was no reduction found
in the group that brushed and flossed. These results showed that flossing does not
immediately improve the health of the oral cavity. Zimmer et al. (2006) suggests this
could be because many people do not know how to floss correctly and therefore are not
doing themselves any good by flossing. The results also showed that mouthwashes in
combination with brushing have a higher plaque reduction than that of brushing in
combination with flossing. The combination of brushing and rinsing with an
antimicrobial mouthwash is not only easier and more often performed correctly than
5
flossing, but it is clinically shown to kill more oral bacteria and reduce plaque more
effectively (Zimmer et al., 2006).
Non-chlorohexidine containing mouthwashes have been found to reduce plaque
and microbial growth without the negative side effects of chlorohexidine solutions (Fine
et al., 1985). Listerine® is a common over-the-counter mouthwash that does not use
chlorohexidine as an active ingredient but instead uses essential oils which are thymol
and menthol. It has been clinically shown to reduce dental plaque and microbial growth
(Fine et al., 1985). Fine et al. (1985) conducted a nine month clinical trial to investigate
the effect of Listerine® on plaque and oral microbial growth. Individuals rinsed twice
daily with Listerine antiseptic and continued their normal oral hygiene procedures.
Plaque and microbial growth were tested and compared to an alcohol control group,
which contained the same percentage of alcohol as Listerine®, but neither of the active
ingredients of thymol and menthol (Fine et al., 1985). Listerine® was found to reduce
oral plaque and antimicrobial growth in comparison to the water control group. Plaque
was reduced in both the Listerine® and alcohol control groups due to the effect that
alcohol alone has on plaque growth; however, the overall reduction was larger in the
Listerine® than the alcohol control group (Fine et al.,1985).
Essential oils have been used for many years as antimicrobial agents (Filoche et
al., 2005). Listerin®e exploits this antimicrobial quality by using thymol and menthol as
its active ingredients (Filoche et al., 2005). Fine et al. (2005) studied the effectiveness of
Listerine® on different species of Streptococci. This study was done in vitro to ensure
that the strains were pure and had not been contaminated. They found that Listerine® was
more effective against mutans species of Streptococci than other species such as S. mitis.
6
Since S. mutans is the main contributor to dental plaque and other oral health diseases, it
has been shown that mouthwashes containing active ingredients of essential oils will lead
to improvements in oral health if used on a regular basis (Fine et al., 2000). Although
chlorohexidine has been used as the gold standard of oral bacterial reduction, its
effectivness against S. mutans specifically has been debated, while over-the-counter
essential oils mouthwashes have been clinically shown to reduce S. mutans levels
specifically (Menendez et al., 2005). A mouthwash combination of 1% chlorohexidine
and 1% thymol called Certivec® has been recently introduced (Filoche et al., 2005), and
it has been suggested that Certivec® is specifically more effective against S. mutans than
Listerine® or chlorohexidine solutions alone (Filoche et al., 2005). Until a
chlorohexidine solution is available over-the-counter in the United States, consumers are
left to choose from an array of over-the-counter mouthwash brands. Studies have shown
that some over-the-counter brands are as effective as chlorohexidine.
Although rinsing with a mouthwash may be easier and less painful than flossing,
there is still a complaint made by patients about mouthwashes. The amounts of alcohol
contained in mouthwashes and the burning sensation they cause have been a deterrent to
regular mouthwash use by many (Sharma et al., 2003). Commercial mouthwash brands
typically use alcohol as a main ingredient to improve the antibacterial effects of the
mouthwashes. To address this complaint of customers, some mouthwash brands have
introduced alcohol-free solutions. Many alcohol-free mouthwashes use cetylpyridinium
chloride (CPC) as an active antibacterial ingredient; it is effective because it is a cationic
surface active agent that is able to absorb the negatively charged phosphates of the
bacteria’s cell membrane (Radford et al., 1997). Radford et al. (1997) conducted a study
7
to see if CPC-containing mouthwashes had an antibacterial effect on the oral flora. The
researchers compared the antibacterial effectiveness of a 0.05% CPC solution and a
placebo. The 132 participants rinsed with 10 ml of a given mouthwash (either CPC or
placebo) for 30 seconds twice daily; once in the morning and once in the evening
(Radford et al., 2005). After the six week study, the researchers found that the CPC
solution did not reduce or increase the amounts of S. mutans in the oral cavities of the
participants. In another study by Witt et al. (2005), a higher concentration of CPC was
used to determine if this change would make a difference in antibacterial effectiveness.
This study used a 0.07% concentration of CPC and conducted both an in vitro and in vivo
study. The in vitro part of the study was conducted on several different oral bacteria
including S. mutans. The CPC solution exhibited 99% bacteria kill for all bacteria tested
(Witt et al., 2005). The in vivo study tested the same concentration of CPC mouthwash
against a positive control (Listerine®) and a negative control (placebo). Participants
rinsed with 20 ml of an assigned mouthwash for 30 seconds in the morning and in the
evening. The results supported the in vitro study by reducing plaque 25% when
compared to the placebo in just 4 days (Witt et al., 2005). These results show the
effectiveness of a CPC solution at a 0.07% concentration.
Alcohol-containing mouthwash has been clinically shown to be an effective
antibacterial agent, but many people are unable to use this product, such as children,
diabetics, alcoholics, and members of certain religious faiths (Radford et al., 1997).
However, these individuals are able to use alcohol-free brands, such as ones that contain
CPC. The effectiveness against S. mutans of both active ingredients has been clinically
shown. Over-the-counter mouthwashes use a variety of active ingredients at different
8
concentrations. The purpose of my study was to examine which of these mouthwash
brands, active ingredients, and concentrations was most effective against S. mutans.
My hypothesis was two-fold due to the two part experiment being conducted.
The first is a comparison of over-the-counter mouthwashes to determine which has the
greatest antimicrobial activity against S. mutans. I believed that Listerine® would exhibit
the highest antimicrobial growth inhibition due to the active ingredients present.
Listerine® contains thymol and menthols as its main active ingredients, and also contains
a high level of alcohol (21.6%). Previous studies have shown that Listerine’s® active
ingredients work together to be highly antimicrobial, while working alone they are less
effective (Fine et al., 1985). Due to these studies, I hypothesized that the highest
experimental concentration of CPC would exhibit higher antimicrobial activity than other
tested active ingredients in the second experiment because CPC is sold commercially in
mouthwashes as the only active ingredient present. This shows that CPC can work
effectively without other active ingredients.
II. Methods
This study evaluated the antimicrobial effectiveness of mouthwashes against S.
mutans. The first test performed was a test of 10 different over-the-counter mouthwashes
compared to a negative control of water. S. mutans was acquired from Ward’s Natural
Science and was spread evenly across Tryptic Soy agar (TSA) Petri dishes with a sterile
glass rod. Filter paper discs were dipped half way into each mouthwash using sterile
tweezers and placed on the pre-inoculated Petri dishes. The Petri dishes were incubated
at a normal body temperature (37ºC) for 24 hours (Fine et al., 2000). This amount of
time allowed the S. mutans to grow enough to form a lawn over the surface. If the Petri
9
dishes had been left in the incubator much longer than 24 hours, then I would have run
the risk of the bacteria using up the nutrients in the agar; once the nutrients had been used
up the bacteria would begin to die (Fine et al., 2000). After 24 hours of incubation, the
zone of inhibition of bacterial growth was measured in millimeters (Al-Masallam et al.,
2004).
In the second test, the specific active ingredients present in the mouthwashes
were purchased from Fisher Scientific Laboratory Supplies or were provided by St.
Martin’s University. Over-the-counter mouthwashes contain different concentrations of
active ingredients. In order to determine the concentration with the highest antimicrobial
effect for each active ingredient, I diluted each stock solution into three experimental
concentrations. S. mutans was spread onto Petri dishes in the same manner as in the first
experiment. I then dipped filter paper discs into each of the diluted concentrations of the
four active ingredients and applied them with sterile tweezers to the Petri dishes
containing the bacteria. The Petri dishes were incubated at 37ºC for 24 hours, and the
zone of inhibition was measured in millimeters to determine the most effective active
ingredient concentration among those tested (Al-Masallam et al., 2004). To make sure
that I had accurate data for analysis, each of the 10 mouthwashes, the water control, and
the 4 active ingredient’s dilutions (3 each) were tested with 20 replicates each. An
average zone of inhibition of the replicates was calculated. Water was used as the control
and was applied to the filter paper disks by dipping them halfway into the water and then
to the Petri dishes contaminated with S. mutans in the same manner as the mouthwashes
and active ingredients.
10
A. Agar Preparation for Petri Dishes
A nutrient agar was made to pour into each Petri dish before the S. mutans was
grown on them. Tryptic Soy agar (TSA) was recommended for anaerobic bacteria such
as S. mutans for maximum growth efficiency (Filioche et al., 2005). The directions for
agar preparation acquired from Ward’s Natural Science recommended 8 grams of agar
powder added to each liter of deionized water. The agar was mixed and heated for 30
minutes with a magnetic stirrer to dissolve the agar in the deionized water (Brown, 2005).
This mixture was placed in a Tuttnauer 2540E autoclave for 15 minutes at 15 pounds per
square inch (psi) and 121ºC to sterilize the agar. An autoclave is a device that uses
pressure to heat liquid solutions above their boiling point to sterilize them (Brown, 2005).
The agar was then cooled for 30 minutes, enough time for it to have cooled down, but not
to have hardened. Approximately 10-12 ml of agar was poured into each of 115 Petri
dishes (Brown, 2005). The Petri dishes were allowed to cool at room temperature until
hardened and placed in the refrigerator upside down to prevent condensation from
forming on the surface of the agar.
B. Growing Streptococcus mutans
A stock colony of the bacteria was made to have a solution of bacteria from which
to work. The S. mutans was obtained from Ward’s Natural Science in a lyophilized state,
in order to begin a culture growth it needed to be rehydrated. To begin this process,
0.5ml of Tryptic soy broth (TSB) was placed in the cryovial containing the lyophilized S.
mutans with a sterile pipette and left for 60 seconds to rehydrate. To mix the S. mutans
with the nutrient broth completely, the pipette was drawn up and down 10 times. A
11
sterile cotton swab was used to inoculate the TSA slant with S. mutans by dipping the
swab in the mixture and streaking it evenly across the agar. The remaining solution
inside the cryovial was placed into a test tube containing TSB with a sterile pipette. The
test tubes were capped loosely and incubated in a VWR Scientific Inc. incubator at 37ºC
for 72 hours (Ward’s Natural Science, 1993).
C. Diluting Active Ingredients
By diluting my stock solutions of active ingredients to specific concentrations, I
was able to determine which test concentration of the different active ingredients is most
effective against S. mutans. I diluted the active ingredients purchased from Fisher
Scientific Laboratories and provided by Saint Martin’s University into specific
concentrations based on the normal ranges of each active ingredient found in over-thecounter mouthwash brands. Alcohol is an active ingredient in high concentrations in the
alcohol-containing mouthwashes tested in this study ranging from 11-21.6%
commercially. In order to determine which concentration had the highest antimicrobial
activity, I created 3 test dilutions to cover high, medium, and low alcohol content. The
stock ethyl-alcohol solution was diluted into 10%, 20%, and 30% concentrations. The
range of cetylpyridinium chloride (CPC) in mouthwashes is 0.07-0.075%. Test solutions
were diluted into 0.05%, 0.07% and 0.10% concentrations. Thymol and menthol come in
the same concentrations respectively in the essential oils-containing mouthwashes;
thymol is available in a 0.064% concentration and menthol a 0.042% concentration
commercially. Thymol was diluted into 0.05%, 0.07%, and 0.10% and menthol into a
0.02%, 0.05%, and 0.10% test concentrations.
12
To dilute the active ingredients into the proper test concentrations, deionized
water was added to the stock active ingredients (Efiok and Eduok, 1993). The exact ratio
of deionized water to add to the corresponding active ingredient was calculated using
equation 1 below (Efiok and Eduok, 1993).
Equation 1.
C1 / C2 = V2 / V1
C1 = Volume of stock reagent
C2 = Concentration of stock reagent
V2 = Final volume needed
V1 = Final Concentration needed
Table 1 shows the amount of each active ingredient added to deionized water to
create the test concentrations of the active ingredients.
13
Table 1. Amount of stock active ingredient added to distilled water to create test
concentrations. Ethyl alcohol was used for the alcohol test groups. The CPC labeled
groups are cetylpridinium chloride. Test concentrations represent actual active
ingredients found commercially in over-the-counter mouthwash brands.
Alcohol
CPC
Thymol
Menthol
10.0% alcohol
20.0% alcohol
30.0% alcohol
1 ml alcohol
+
9 ml H2O
2 ml alcohol
+
8 ml H2O
3 ml alcohol
+
7 ml H2O
0.05% CPC
0.07% CPC
0.10% CPC
0.25 ml CPC
+
499.75 ml H2O
0.25 ml CPC
+
354.75 ml H2O
0.10 ml CPC
+
99.9 ml H2O
0.05% thymol
0.07% thymol
0.10% thymol
0.25 ml thymol
+
499.75 ml H2O
0.25 ml thymol
+
354.75 ml H2O
0.10 ml thymol
+
99.9 ml H2O
0.02% menthol
0.05% menthol
0.10% menthol
0.25 ml menthol
+
1249.75 ml H2O
0.25 ml menthol
+
499.75 ml H2O
0.10 ml menthol
+
99.9 ml H2O
14
D. Applying Mouthwash, Active Ingredient Dilutions, or water control to Petri
Dishes Containing Streptococcus mutans
The mouthwashes, active ingredients, and control (water) were applied to filter
paper discs; each Petri dish was used for 4 filter paper discs. The average zone of
inhibition of bacterial growth was measured and statistically analyzed. The mouthwashes
were divided into three groups based on their active ingredients, which were alcohol,
CPC, and essential oils (thymol and menthol). Tables 2-4 below show the brand names
and manufacturing information for each mouthwash tested.
Table 2. Part A shows the alcohol-containing mouthwashes tested during this study. The
brand name of the mouthwash is located in the left column, while the manufacturer and
location are located in the right column. Part B shows the cetylpridinium chloridecontaining mouthwashes tested during this study. The brand name of the mouthwash is
located in the left column, while the manufacturer and location are located in the right
column. Part C shows the essential oils-containing mouthwashes tested during this study.
The brand name of the mouthwash is located in the left column, while the manufacturer
and location are located in the right column.
Brand
Manufacturer
A.
Dr. Tichenor’s®
Plax®
Targon®
Dr. G.H. Tichenor’s Co., New Orleans, LA
Pfizer Consumer Healthcare, Morris Plains,
NJ
GlaxoSmithKline, Moon Township, PA
B.
Crest Prohealth®
Breath Rx®
Walgreen’s Fluoride Rinse®
Viadent®
Scope®
Proctor & Gamble, Cincinnati, OH
Discus Dental Inc., Culver City, CA
Walgreen Co., Deerfield, IL
Colgate Oral Pharmaceuticals Inc., Canton,
MA
Proctor & Gamble, Cincinnati OH
C.
Listerine®
Walgreen’s Fresh Breath®
Pfizer Consumer Healthcare, Morris Plains,
NJ
Walgreen Co., Deerfield, IL
15
The zone of inhibition test was performed by dipping sterilized filter paper discs
(sterilized using a Tuttnauer 2540E autoclave) into 10 ml of each of the 10 mouthwash
brands, 4 active ingredient dilutions (3 each), or control (water) half way to the top of the
disk using sterile forceps. Twenty separate filter paper discs were used for each
treatment. The filter paper discs were then applied to sterile Petri dishes pre-inoculated
with S. mutans. The Petri dishes were pre-inoculated using a micropipette to pipette 250
μL of S. mutans onto each Petri dish. This allowed bacterial growth to be inhibited in a
circular pattern around each disk. Four discs were placed in quadrant form equal distance
away from each other and the edge of the Petri dish using sterile forceps. This procedure
produced 20 filter paper discs applied to 5 Petri dishes for each treatment. Each of the
115 Petri dishes were incubated at 37° C for 24 hours before the zone of inhibition was
measured for statistical analysis (Al-Masallam et al., 2004). The ring of no bacterial
growth was visible to the naked eye and was measured to the nearest millimeter using a
ruler (Brown, 2005). These rings of inhibition were no uniformly circular, so 3 diameter
measurements were taken for each zone of inhibition and averaged (Al-Masallam et al.,
2004).
E. Statistical Analysis
An analysis of variance (ANOVA) statistical test was performed on the data
collected from the zone of inhibition test (Al-Masallam et al., 2004). There were two
different experiments analyzed; the first is the bacterial zone of inhibition data from the
10 mouthwash brands and control. The average zone of inhibition was calculated for
each of the 10 mouthwashes and for the control. This data was compared using an
ANOVA test. The second statistical analysis compared the data collected from the 4
16
active ingredient dilutions and the control. The average zone of inhibition was calculated
for the active ingredient’s dilutions. The same control was used from the first experiment.
Two one-way ANOVA tests were performed on the data collected, one ANOVA test for
the zone of inhibition data from the mouthwashes and one for the zone of inhibition data
from the active ingredients. Two Tukey multiple comparisons test were performed using
Minitab® (Minitab®, 2005) to determine the significant differences (p-value < 0.05)
between specific mouthwashes and between specific active ingredient dilutions (Heath,
1994).
III. Results
The results were obtained by conducting zone of inhibition tests of 10 over-thecounter mouthwash brands, and 12 mouthwash active ingredient dilutions tested for the
antimicrobial effects on Streptococcus mutans. The control in this study was distilled
water with an average zone of inhibition of zero.
Figure 1 displays the average zone of inhibition measured in millimeters for 10
different over-the-counter mouthwash brands. Over-the-counter mouthwash brands were
compared using an ANOVA statistical test and a Tukey multiple comparisons test
(Minitab®, 2005). There was a significant difference found by the ANOVA statistical
analysis between the average zone of inhibitions of the 10 over-the-counter mouthwash
brands (F = 35.49, d.f. = 9, p-value = 0.0005). There were large variations in sizes of
zone of inhibition between the mouthwashes; they ranged from Walgreen’s Fresh
Breath® at 0.0mm to Scope® at 14.6mm. Scope® exhibited the highest average zone of
inhibition, and was found to be significantly higher than Targon®, Dr.Tichenor’s®, Breath
Rx®, Plax®, Walgreen’s Fresh Breath® and Viadent® with a 95% confidence interval.
17
Walgreen’s Fresh Breath® exhibited an average zone of inhibition of zero, and was found
to be significantly lower than all other mouthwash brands tested.
16
14
12
10
8
6
4
2
Via
den
t
sh B
rea
th
Fre
n's
ree
Bre
ath
Rx
Pla
x
Wa
lg
Wa
lg
Dr.
ree
n
's F
Tich
e
lou
ride
nor
's
alth
st P
roh
e
Cre
Tar
gon
Sco
pe
List
erin
e
0
Figure 1. Average zone of inhibition of bacterial growth of 10 different over-the-counter mouthwashes
measured in millimeters including standard error bars which represent one standard deviation from the
mean for 20 replicates per mouthwash brand. Walgreen’s Fresh Breath exhibited an average zone of
inhibition of zero millimeters.
Figure 2 displays the average zone of inhibition including standard errors for
each of the 12 active ingredient dilutions tested. The average zone of inhibition the
alcohol test groups ranged from 5.0-7.2mm, the CPC test groups ranged from 9.210.4mm, and the menthol test groups ranged from 4.3-5.8mm and the thymol test groups
ranged from 0-0.3mm (Fig. 2).
18
12
10
Zone of Inhibition (mm)
8
6
4
2
0
10%
Alchohol
20%
Alcohol
30%
Alchohol
0.05%
CPC
0.07%
CPC
0.10%
CPC
0.05%
Thymol
0.07%
Thymol
0.10%
Thymol
0.02%
Menthol
0.05%
Menthol
0.10%
Menthol
Figure 2. Average zone of inhibition of bacterial growth of mouthwash active ingredient dilutions
measured in millimeters for 20 replicates per test concentration. The error bars represent one standard error
from the mean. Columns labeled with CPC represent cetylpridinium chloride solutions. The 0.05% and
0.10% thymol concentrations each exhibited average zones of inhibitions of zero.
The ANOVA statistical test showed a statistical difference among the different
over-the-counter mouthwash brands ( F = 34.63, df = 11, p-value 0.0005). The active
ingredient dilutions with the largest zone of inhibitions were 0.07% and 0.10% CPC; they
were found to be significantly higher than all menthol and thymol dilutions by a Tukey
multiple comparisons test with a 95% confidence interval. The active ingredient
dilutions with the smallest zone of inhibitions were 0.05% and 0.10% thymol. These
were both found to be significantly lower than all of alcohol, CPC, and menthol dilutions.
IV. Discussion
Previous studies investigating the antimicrobial effectiveness of over-the-counter
mouthwashes on S. mutans found that Listerine®, because of its essential oil ingredients
(thymol and menthol), is effective to use in place of such prescription mouthwashes as
19
chlorohexidine (Fine et al., 2000). My research contradicts these studies because, I found
that Scope®, Crest Pro-Health® and other CPC containing mouthwashes inhibit S. mutans
growth more than Listerine® (F = 35.49, d.f. = 9, p-value = 0.0005). However, Listerine®
was shown to be effective against S. mutans, although inhibiting less growth than the
CPC containing mouthwashes.
Scope® and Crest Pro-Health® had the highest growth inhibition of S. mutans.
Crest Pro-Health® is alcohol-free and contains 0.07% CPC, while Scope contains 15%
alcohol and 0.07% CPC as its active ingredients. There was no significant difference (pvalue = 0.0005) between the zones of inhibitions of the two mouthwashes. My active
ingredient dilution data supported these findings, as the 0.07% CPC dilution exhibited a
significantly higher growth inhibition (p-value = 0.0005) than the other active ingredients
tested, except for the 0.10% CPC solution. The 0.10% CPC solution exhibited a slightly
lower inhibition than the 0.07% CPC, but was not significantly different. Further
analysis of the active ingredients of Listerine® showed that both the essential oils, thymol
and menthol, inhibited growth significantly less (p-value = 0.0005) than CPC. Thymol,
in its 0.05% and 0.10% dilutions, showed no inhibition of bacteria growth, while the
0.07% dilution showed very little, with an average zone of inhibition of just 0.3 mm. The
menthol dilutions exhibited more growth inhibition, but the different concentrations were
not significantly different from each other or from the 10% alcohol dilution (p-value =
0.0005). The combination of essential oils and alcohol had a greater inhibitory effect on
S. mutans growth than either ingredient alone; this is the likely reason for the greater
inhibitory effect by Listerine® than the active ingredients alone. Although no significant
difference (p-value = 0.0005) was found between Scope® and Crest Pro-Health®, I
20
recommend the use of Crest Pro-Health® because it contains no alcohol and is therefore
suitable for a larger range of individuals. Many people are unable to use alcoholcontaining mouthwashes such as children, diabetics, alcoholics, and members of certain
religious faiths (Radford et al., 1997), and individuals with everyday concerns of the
burning sensation of the mouth due to alcohol. These results support previous studies
conducted on Crest Pro-Health® that showed it had high antimicrobial capabilities
without the use of alcohol (Witt et al., 2005).
Small variations of S. mutans inhibition were found within each test group,
showing that increase of replicates should not show differences among the data recorded;
therefore increasing the replicate size is not likely to change the average zone of
inhibition data for the mouthwashes or the active ingredient dilutions. In vivo studies
would be needed to examine the antimicrobial effectiveness of these over-the-counter
mouthwashes in the human oral environment. The presence of different strains of S.
mutans and other types of oral bacteria may show differences in the growth inhibition
effectiveness because the mouthwashes may not react in the same way. The oral
environment varies considerably from person to person; an in vivo study could examine
how the individuals’ diet, genetics, and personal hygiene techniques may alter the
effectiveness of over-the-counter mouthwashes from their effectiveness shown in vitro.
Expanding the number of different mouthwashes and active ingredients tested would give
additional information. The active ingredients I selected to test were based on the four
that are most commonly used over-the-counter, and because they typically came in the
highest percentages compared to other active ingredients. Some other active ingredients
that are common in the mouthwashes tested, but that I did not examine individually are
21
eucalyptol, hexetidine, methyl salicylate, benzalkonium chloride, methylparaben,
hydrogen peroxide, and fluoride. By expanding the mouthwashes and active ingredients
tested, a larger view upon the trends of antibacterial effectiveness against S. mutans
would be shown. According to the American Dental Administration, Listerine® is
currently the world’s top selling over-the-counter mouthwash, it contains other active
ingredients besides the alcohol and essential oils that I tested, it contains eucalyptol and
methyl salicylate. These active ingredients may be the reason why Listerine® is shown to
be more effective than in its individual active ingredients.
The bacteria present in plaque, such as S. mutans, are able to turn the
carbohydrates that we ingest into acids which demineralizes the teeth, leading to tooth
decay. Over-the-counter mouthwashes have the ability to provide an easy everyday
solution for many oral diseases such as dental caries (cavities) and gingivitis (Scherp,
1971). They are able to reduce the amount of harmful bacteria present in the oral cavity
by reducing their growth to prevent the buildup of plaque on the surface of the teeth.
Cavities are costly and painful, but are avoidable with proper oral hygiene techniques
such as brushing and flossing daily. Mouthwashes are able to provide an antibacterial
influence to help further reduce the amount of harmful bacteria in the mouth.
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V. Acknowledgments
I would like to thank Dr. Mary Jo Hartman and Dr. Margaret Olney for their
diligent editing and mentoring throughout my research project. I would like to thank my
biology senior seminar colleagues for their peer reviews and constant support throughout
this year. Finally, my lab work could never have been accomplished with such ease
without the help of Cheryl Guglielmo who volunteered many hours helping me collect all
of the required materials for my research.
23
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