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A Quantitative Comparison of the Zone of Inhibition against Streptococcus mutans
and Oral Bacterial Growth using Crest®, Colgate®, and Aquafresh® Toothpastes
Final Draft
Kevan Johanson
Senior Seminar 2
5-8-2007
1
Table of Contents
Page Numbers
1
1. Abstract &
2. Introduction
2
2. Methods
7
3. Results
13
4. Discussion
16
5. Consent Form – Appendix 1
19
6. Acknowledgements
21
7. Literature Cited
22
2
Abstract
This study evaluated the antibacterial efficiency of Crest®, Colgate®, and
Aquafresh® toothpastes using in vivo and in vitro experiments. The in vivo experiment
measured the optical density of bacteria samples grown from participants’ teeth by using
a spectrophotometer. The in vitro experiment measured the zone of inhibition of the
three toothpastes against Streptococcus mutans, with water as a control group. Statistical
analyses showed no difference in optical density among Crest®, Colgate®, and
Aquafresh® toothpastes. However, there was a significantly larger zone of inhibition for
the Colgate toothpaste compared to Crest®, Aquafresh, and control groups (p = 0.0005 at
a 98.95% confidence interval).
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Introduction
Dental hygiene is an important field in human health. Pathogenic and nonpathogenic bacteria are constant residents of the human mouth. Scientific research has
shown that the use of oral hygienic products plays a vital role in the reduction of human
oral bacteria. One of toothpaste’s purposes is to prevent oral bacteria growth, in order to
prevent pathogenic infection. Within the oral cavity, the most common cariogenic
(caries-causing) of the Streptococci genus is Streptococcus mutans (Tortora et al., 2004).
My study observed the antibacterial effectiveness of Colgate®, Crest®, and Aquafresh®
toothpastes on oral bacteria and S. mutans. The reason I chose toothpaste for this study
was because of its extensive use by humans, many varieties and brands, and easy
availability. The overall purpose of most active ingredients of toothpaste is to maintain
healthy teeth and gums by preventing bacterial growth in the oral cavity. The active
ingredients of toothpastes can include antibacterial agents, anti-cavity agents, enzymes
for amplifying saliva’s antibacterial properties, and enzymes that prevent decay. The
antibacterial agent triclosan and the anti-caries (cavity) agent fluoride are common
ingredients in toothpaste. For my study, I examined the antibacterial activity of triclosan
and fluoride in three toothpastes.
Sullivan et al. (2003) tested toothpaste containing triclosan on resistant oral
Streptococci and measured the in vitro sensitivity of Streptococci strains against triclosan.
Triclosan is an antibacterial agent commonly found in soaps, mouthwash, toothpaste, and
other hygiene items. Triclosan is a biphenolic derivative that kills gram positive bacteria
such as Streptococci and Staphylococci. A biphenol is a derivative of phenol, which
either helps reduce irritation or increases antibacterial activity. Triclosan inhibits
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enzymes that synthesize fatty acids, which compromise the bacterium’s plasma
membrane (Brown, 2005). The Sullivan et al. (2003) study also analyzed the impact of
triclosan on the normal oral microflora. Nine subjects used toothpaste containing 0.3%
triclosan and sodium lauryl sulphate to brush their teeth twice a day for fourteen days.
The nine subjects were in good health, and none had been administered antibacterial
agents in the three months before the study. On the first day of the study, 5 saliva
samples in 15 minutes were collected per subject. The concentration of the triclosan in
saliva was 3.6 g/mL of saliva, which was determined by agar diffusion procedures
(Sullivan et al. 2003). Once the saliva concentration had been determined, the saliva was
added to and incubated on selective agar plates. Streptococci strains were grown into
colonies on the plates and subsequently isolated into pure cultures by T-square methods.
The purpose of the T-square technique was to isolate bacteria from mixed species
colonies down to the genus level (Brown, 2005). Gram stain and biochemical tests were
conducted to identify the bacterial species and colony forming units (CFU). As a result
of triclosan use, there were reductions of Lactobacilli in the oral microflora; however,
there was no change in the susceptibility of Streptococci towards triclosan. Lactobacilli
showed a significant reduction in concentration from 5.5 CFU on day 0 to 5.2 CFU on
day 14 with a p-value of less than 0.05 using an Analysis of Variance (ANOVA). S.
mutans showed no increased susceptibility to triclosan. Sullivan et al. (2003) reported
3.4 CFU for S. mutans on day 0 and a CFU of 4.1 on day 14. Data analysis determined
that using triclosan in toothpaste showed no statistically significant difference on the in
vitro sensitivity of resistant oral Streptococci. My study observed the antibacterial
activity of three toothpastes on S. mutans by measuring zones of inhibition. The study by
5
Sullivan et al. (2003) influenced this study to test the effectiveness of triclosan in
toothpaste against S. mutans and oral bacteria.
Battino et al. (2004) researched the in vitro antioxidant activity of 12 antioxidantenriched toothpastes. Antioxidants in toothpaste may benefit the host’s antioxidant
defenses in the case of periodontal diseases (PD). PD are chronic inflammatory
conditions that arise from the interaction between pathogenic bacteria and the host
immune responses. The toothpastes contained mixtures of sodium ascorbyl phosphate,
alpha-tocopherol acetate, pycnogenol, allantoin, and methyl salycilate. The antioxidant
activity was measured by a series of tests, such as, the ABTS-decolorization assay and
the Comet assay (Battino et al., 2004). The results indicated that antioxidant activity
depended on the solubility of the active ingredients. The toothpastes that contained
sodium ascorbyl phosphate displayed clear I50 values (mg toothpaste / ml water) ranging
from 50 to 80 mg of toothpaste per milliliter of water, which measure antioxidant
concentration. The Comet assay showed limited antioxidant protection from sodium
ascorbyl phosphate when the toothpaste was in the presence of hydrogen perioxide.
However, Battino et al. (2004) found that sodium ascorbyl phosphate in toothpaste
possessed antioxidant activity. This study was similar to mine; because I tested three
toothpastes for their antibacterial properties as Battino et al. (2004) did with antioxidant
properties.
De Leo et al. (1990) researched the prevalence of S. mutans and dental decay in
children from Genoa, Italy. This study’s goal was to obtain information on the
prevalence of dental decay and to find the proportional amount of Streptococcus mutans
in plaque samples from subjects. Samples of saliva, bacteria, and plaque from the first
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molar in each quadrant were collected. After the saliva was spread on selective agar
plates for growth, S. mutans in the samples was reported as present or absent. De Leo et
al. (1990) reported S. mutans was present in 58 of the 105 subjects. Seventy-seven of the
subjects were caries active (decayed teeth) and 28 subjects were caries-free. The
presence of S. mutans in subjects with caries was 63%. S. mutans was found in only 31%
of subjects with no caries. S. mutans was associated with dental decay, by their levels in
plaque samples.
Clayton et al. (2000) tested the effects of penicillin on Endamoeba gingivalis
from oral bacterial cultures (Clayton et al., 2000). They obtained E. gingivalis from each
subject’s mouth. The bacteria were spread onto warmed culture media. After
incubation, the bacteria displayed no decrease in stability. The penicillin concentrate had
little or no effect on bacterial growth, because the pH remained at the bacterium’s
optimum level. Clayton et al. (2000) ultimately found that their prescribed penicillin
dosage was not enough to kill the bacteria in the log and lag phases of growth. This
article brought to my attention that researching toothpastes’ ingredients was critical for
testing their antibacterial efficacy against oral bacteria. I used well-known name-brand
toothpastes containing the active ingredients triclosan and fluoride.
Romao et al. (1990) conducted tests on the effectiveness of human saliva as a
cleaning agent. The use of saliva for cleaning has been practiced for centuries (Romao et
al., 1990). They conducted solubility and resistance tests to measure the effect of saliva
on different surfaces. By using thin-layer chromatography to analyze the lipids in dirt
that had been removed by salvia (Romao et al. 1990), they found that saliva removed
lipids from the dirt. The results reported that saliva was an effective surface cleaner.
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One concern was that the saliva degraded the surface of the object and attacked red and
blue pigments, vermilion and azurite respectively. The vermilion and azurite colors were
degraded due to the enzymatic activity of saliva. Their study was significant because
saliva plays an important role in cleaning the human mouth. However, since saliva was
nearly impossible to control in my study, I assumed it was constant among all subjects.
I compared the antibacterial activities of three toothpastes: Crest®, Colgate®,
and Aquafresh®. Crest®, Colgate®, and Aquafresh® toothpastes all contained sodium
fluoride for anti-cavity protection. Colgate® was the only toothpaste I used that
contained triclosan. I had two separate hypotheses: one for the in vivo experiment and
the other for the in vitro experiment. First, I hypothesized that bacterial growth would be
the most inhibited by Colgate® toothpaste with a p-value less than 0.05, which would
have had a lower optical density than the samples treated with Crest® and Aquafresh®
toothpastes. I predicted triclosan and sodium fluoride together would be more effective
in killing oral bacteria than sodium fluoride alone. The second hypothesis was Crest®,
Colgate®, and Aquafresh® would show no significant difference in zone of inhibition
measurements of S. mutans. This prediction of no difference between the different
toothpastes’ antibacterial activity was because the in vitro sensitivity of Streptococcus
mutans was resistant towards triclosan according to Sullivan et al. (2003). The data from
the two experiments in this study were analyzed using ANOVA and Tukey multiple
comparisons tests (Minitab®, 2005), to quantitatively compare antibacterial efficacy
among the three toothpastes. This study provided statistical evidence regarding these
toothpastes’ antibacterial efficacy in vivo and against S. mutans in vitro.
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Methods
I conducted two different experiments with three toothpastes. The methods are
presented separately for the two experiments and are independent of each other. The in
vivo experiment tested the amount of oral bacteria present in subject after using
toothpaste. Subjects participated by using toothpaste for a week. The swabbing samples
collected on days 1 and 8, before and after brushing their teeth were incubated in nutrient
broth and measured for absorbance using a spectrophotometer. The in vitro experiment
measured the zone of inhibition from the toothpastes against S. mutans. After spreading
a bacterial lawn, I applied filter discs from toothpaste slurries to the plates and incubated
for 24 hours at 37°C. The zone of inhibition was conducted to determine the antibacterial
efficacy of toothpaste in preventing growth of S. mutans.. During the experiments, I
always wore goggles, gloves and lab coat and used sterile cotton swabs to protect the
health of the participants and myself, along with maintaining a sterile environment to
avoid contamination.
Subject Experiment using Toothpaste
For the first experiment using human subjects, I analyzed a total of 30 subjects
divided evenly among three toothpastes. No subjects were used who were participating
in oral studies that would drastically influence my studies’ aim. The subjects in the
mouthwash study were valid as participants in my study because mouthwash is a regular
hygienic task. The subjects were at least 18 years of age and recruited from Saint
Martin’s University. Consent forms were approved by the Institutional Review Board
(IRB), which reviews proposed studies using human subjects before approving them to
use subjects ( Appendix 1), signed by each subject, and placed in a sealed envelope
9
before any testing took place. Each subject was then evaluated from a questionnaire, in
order to distribute the tobacco users evenly amongst the three tested toothpastes. The
subjects used one of the three following toothpastes: Crest®, Colgate®, and Aquafresh®.
I selected these toothpastes because they were similar when comparing their active
ingredient, sodium fluoride (Battino et al., 2005) and because they are commonly used by
consumers. I tested Colgate® because it additionally contained the antibacterial phenol,
triclosan. The subjects were given toothbrushes and toothpaste and required to brush
twice daily for 7 days. I dispensed the toothpastes into pill containers because it kept the
toothpaste clean and it made it easy for subjects to use. I provided subjects with their
assigned toothpaste, gave them new toothbrushes, and provided directions with contact
information if they had any questions.
I swabbed each participant’s mouth twice on day 1 and 8 of the study. After
subjects rinsed their mouth with water for 30 seconds, the first swab for bacteria was
collected from their mouth. The subjects then brushed their teeth for two minutes and
rinsed with water before a second swab was collected in the same area. Each time I
collected bacteria, I thoroughly swabbed near the crown and gum-line area of the front of
mandibular (bottom) teeth to ensure collection. The sterile swabbing precautions I used
were important to maintain the safety and health of the subjects and researcher. Each
swab was transferred to its own nutrient broth tube to incubate at 37˚C for 24 and 48
hours.
Preparation of Nutrient Broth Tubes
In preparation for the human experiment, I prepared 120 nutrient broth tubes (4
broth tubes per subject) by adding about 10 mL to each tube totaling 1.2 liters. I mixed
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9.6 grams of Ward’s® nutrient broth powder with 1.2 liters of deionized water in an
Erlenmeyer flask. Once the broth was completely dissolved and poured into tubes, the
tube caps were loosely screwed on to be autoclaved. The tubes were sterilized in the
Tuttnauer 2540E autoclave at 121°C and 15 psi for 15 minutes, tightly capped with
labels, and placed in the refrigerator at 4°C.
Using the Spectrophotometer
The swabs from days 1 and 7 were submerged in separate nutrient broth tubes and
incubated for growth at 37°C for 24 and 48 hours. After the broth was incubated for
bacterial growth, a sample of each tube was transferred into individual cuvettes to prepare
for the Spectronic Instruments D20+ spectrophotometer. The spectrophotometer reads
the optical density of bacteria by measuring absorbance of light through the broth
(Brown, 2005). The absorbance data were collected from the broth sample after the 24th
and 48th hour of incubation. After the spectrophotometer had warmed up for 10 minutes,
I tuned the wavelength to 686 and set the absorbance to zero (Brown, 2005). I then
poured the sterile nutrient broth control into a cuvette and reset the absorbance at zero.
The cuvette samples were placed in the spectrophotometer and absorbance data were
recorded. The growth period of 24 hours was selected because it mimics brushing teeth
once per day. The temperature during incubation was 37°C, because this is the optimum
temperature for most bacteria in humans and the average temperature of the human body.
The optical density measurements allowed me to compare the antibacterial activity of the
three toothpastes. On day 1, I measured the instant impact of the toothpaste and on day 7
the data allowed me to compare the antibacterial activity of the three toothpastes after
short-term use.
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Lab Experiment using Toothpaste
For testing the zone of inhibition, I prepared 10 Petri plates for each of the
Crest®, Colgate®, and Aquafresh® toothpastes. The experiment required cultured
Streptococcus mutans, Ward’s® Tryptic Soy Agar plates, and prepared filter discs. The
plates for each toothpaste were compared after measuring the zone of inhibition for data
analysis.
Preparation of Nutrient Agar Plates
For the 30 Petri plates testing zone of inhibition, I calculated I would use 360 ml
of Ward’s® Tryptic Soy Agar (TSA), which is 12 ml for each Petri plate. I dissolved
14.4 grams of Tryptic Soy Agar powder into 360 ml of deionized water in an Erlenmeyer
flask. I placed foil around the top of the flask and autoclaved at 121°C and 15 psi for 15
minutes. Approximately 10 to 12 ml of Tryptic Soy Agar was poured onto each plate and
allowed to cool until solidified. Until the toothpaste slurries and S. mutans cultures were
used for zone of inhibition, I labeled agar plates and refrigerated them at 4°C.
Growing Cultures of Streptococcus mutans
S. mutans was grown with a Rehydration of Cultures package from Ward’s®
Lyophilized Bacteria and Fungi (Ward’s, 1993). The cultures of S. mutans were made by
pipetting 0.5 mL of Ward’s® Trypticase Soy Broth into the cryovial of bacteria granules.
The bacteria solution was then pipetted in and out to allow bacteria pellets to soften for a
minute. The solution was pipetted into the sterile Ward’s® Trypticase Soy Broth tube
provided in the culture kit. This first generation culture was incubated at 37°C for 24
hours and grown for future cultures (Ward’s, 1993). I made 4 BBL® Trypticase Soy
Broth (TSB) tubes for more cultures by dissolving 1.5 grams of the broth powder with 50
12
ml of deionized water. I then poured the broth into the tubes, capped them loosely, and
autoclaved at 121°C and 15 psi for 15 minutes. After I allowed the BBL® TSB tubes to
cool, I used aseptic technique to inoculate the tubes with the S. mutans from the Ward’s®
TSB tube. I conducted these inoculating procedures for the 4 BBL® TBS tubes and
incubated them for 24 hours at 37°C resulting in S. mutans cultures.
Making the Toothpaste Slurries
To prepare the toothpaste for the zone of inhibition, I made a water-based solution
containing a specified amount of toothpaste. Before saturating the filter discs, I
autoclaved Petri plates and deionized water to ensure sterile supplies. I poured 3 mL of
water into the plate and added 1.5 grams of weighed toothpaste (Leyster, 2006). I stirred
the mixture with a sterile stirring rod until homogenous. The toothpaste slurries were
subsequently applied to plates for the zone of inhibition.
Testing the Zone of Inhibition
To start the experiment of the zone of inhibition, I transferred S. mutans to
Tryptic Soy Agar by inoculating and spreading the bacteria from the BBL® TBS tubes
onto the entire agar surface of the 35 plates with a sterile bent glass rod. I prepared
toothpaste slurries for the filter paper before spreading bacteria on plates. I autoclaved
distilled water to sterilize the slurry and added 2 ml of water for every 1 gram of
toothpaste in a sterile beaker. After stirring the mixtures, I used sterile forceps to saturate
the filter discs in the slurry and placed each disc onto the plate’s quadrants (Brown,
2005). Thirty plates, ten for each toothpaste, contained only one designated brand of
toothpaste with 2 filter paper discs per plate. I tested the zone of inhibition on 5
additional S. mutans plates with sterile water as a control for data analysis. The plates
13
were turned upside down to prevent condensation and incubated for 24 hours at 37°C for
growth (Sullivan et al., 2003). I measured the zones of inhibited bacterial growth with a
ruler after the 24 hour mark of incubation. The zone of inhibition was measured by the
diameter in millimeters.
Data Analysis
The data collected from these two experiments were analyzed with statistical
tests. Since both experiments (in vivo and in vitro) have three different groups of
toothpaste, I used a one-way Analysis of Variance (ANOVA) test to compare the zones
of inhibition and bacterial growth for each group. I used Minitab® (2005) for the
ANOVA and Tukey tests. My goal for the subject experiment was to compare the three
toothpastes for their antibacterial properties from quantitative data. My aim for the zone
of inhibition was to see if there was a statistically significant difference amongst the zone
of the three toothpastes and control using a one-way ANOVA. When the ANOVA
showed a statistically significant difference at alpha equals 0.05, I performed Tukey
multiple comparisons tests using a 95% confidence interval.
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Results
Subject Experiment using Toothpaste
After measuring the samples in the spectrophotometer, I compared data among
the three toothpastes for each time bacteria were collected. The samples collected with
high turbidity of bacterial growth showed a higher optical density, and lower optical
density samples had relatively lower bacterial growth. An ANOVA showed no
difference in absorbance from the three toothpastes for each bacteria collection (p >
0.05). The subjects’ measurements from before brushing on day 1 were analyzed using a
one-way ANOVA (Minitab®, 2005), which showed no difference after 24 hours
incubation (F = 0.78; d.f. = 2; p = 0.468). Day 1 after brushing data were then also
compared amongst the three toothpastes with a one-way ANOVA after 24 hours
incubation, which found no statistically significant difference (F = 2.32; d.f. = 2; p =
0.116). An ANOVA test also showed no difference on day 8 before or after brushing
amongst three toothpastes. Since p > 0.05, my hypothesis (significantly lower
absorbance for Colgate users from other toothpaste groups) was rejected. I also
conducted a one-way ANOVA test on the samples from all 32 subjects comparing
bacterial samples before brushing on day 1 to after brushing on day 8, which showed no
statistically significant difference (F = 1.96; d.f. = 2; p = 0.166). In Figure 1, the means
and standard error are represented for all subject samples before and after brushing on
days 1 and 8.
15
0.14
0.12
Absorbance
0.1
0.08
0.06
0.04
0.02
0
before day 1
after day 1
before day 8
after day 8
Figure 1. Absorbance of oral bacteria in broth from incubated swabs taken before and after brushing on
days 1 and 8. Each bar represents the mean and standard error of all 32 subjects for different bacteria
collections. The samples collected before and after brushing are represented as “pre” and “post” for days 1
and 8. Although there was no significant difference between days 1 and 8, this bar graph displays the
optical density absorbance for all subjects for each sample collection after 24 hours incubation.
Lab Experiment using Toothpaste
Once the measurements for the zone of inhibition had been compiled, I analyzed
the data for the three toothpastes and the control using a one-way ANOVA test, which
showed a statistically significant difference amongst the three toothpastes and the control
(F = 105.07; d.f. = 3; P = 0.005) in Figure 2. Using a Tukey multiple comparisons test at
a 98.95% confidence interval, the Colgate toothpaste showed a significantly larger zone
of inhibition compared to Crest, Aquafresh, and control groups. The control showed a
significantly smaller zone of inhibition in contrast to all toothpaste groups (p = 0.0005 @
98.95% confidence interval).
16
50
45
millimeters (mm)
40
35
30
25
20
15
10
5
0
Aquafresh
Crest
Colgate
Control (water)
Figure 2. This chart shows the means of the zone of inhibition for each of the toothpastes. Each
error bar represents one standard error about the mean. The zone of inhibition was measured by
millimeters. The sample sizes were as follows: 19 Aquafresh, 20 Crest, 14 Colgate, and 5 control samples.
While Aquafresh and Crest results showed similar data points, An ANOVA test and Tukey’s multiple
comparisons test at a 95% confidence interval showed the Colgate group had a significantly larger zone
than the Crest, Aquafresh, and control groups. The control group displayed a smaller zone compared to the
Crest, Aquafresh, and Colgate groups.
17
Discussion
For the subject experiment, results showed that no particular toothpaste group had
significantly lower or higher bacteria levels when compared to the other toothpaste
groups. Since, I had hypothesized that Colgate® would have a significantly lower
absorbance than Crest® and Aquafresh® toothpaste groups; I rejected the hypothesis
because data did not support one toothpaste more than another. I had generated my
hypothesis because fluoride and triclosan are found in Colgate®, while the other
toothpastes only had fluoride for an active ingredient, which could have had a significant
influence on toothpaste efficacy. However, I accepted the null hypothesis because there
was no significant difference amongst the toothpastes for repressing bacterial growth (p >
0.05) and there was no evidence that one specific toothpaste was more effective
compared to the others against human oral bacteria.
In future studies of human subjects, it would still be possible that there could be a
significant difference amongst the antibacterial efficiency of the toothpastes I tested. I
would recommend sampling specific teeth and using more precise swabbing techniques
(Mychal Hendrickson, personal communication, April 2007). These swabbing
procedures would be more consistent in the location and technique for bacteria collection.
I also did not invert the broth samples before pouring them into cuvettes. Brown (2005)
suggests inverting and shaking the broths before pouring dilutions to ensure uniform
concentrations of bacterial growth throughout an entire broth sample. Inverting the broth
samples would result in uniform distribution of bacterial growth, and more accurate
absorbance measurements. I would also increase the number of subjects to thirty per
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toothpaste group for this study because it would provide more data to the experiment’s
findings.
If I conducted the subject experiment again, I would sample each subject’s saliva
in addition to the oral study. The bacteria samples collected from subjects would have
contained active ingredients, if saliva was collected during those samples (Burt, 1983).
The different active ingredients of toothpaste and their concentrations in saliva would
have had an impact on the oral bacteria of humans (Sullivan et al., 2003). The active
ingredients can either physically adhere to teeth and gums, or be suspended in saliva. If I
further explored antibacterial efficacy of toothpaste, I would like to test non-fluoride
containing toothpaste, such as Tom’s Toothpaste®.
For the lab experiment testing zone of inhibition, the Colgate toothpaste showed a
significantly larger zone against S. mutans than the Crest, Aquafresh, and control groups
(p < 0.0005 at 98.95% confidence interval). The control of water also displayed a
significantly smaller zone than the toothpaste groups (p < 0.0005 at 98.95% confidence
interval). I hypothesized that there would be no statistically significant difference in the
zone of inhibition of S. mutans amongst the toothpastes. I rejected my hypothesis
because S. mutans showed more resistance towards Crest® and Aquafresh® toothpaste
when compared to the triclosan-containing Colgate® toothpaste from zone of inhibition
experiments (Sullivan et al., 2003).
I did not expect the triclosan-containing Colgate to show larger zones of
inhibition against S. mutans. Many factors in the methods could be attrituted to the data
found. I observed the toothpaste slurry prepared for Colgate® was a viscous liquid
unlike the Crest® and Aquafresh® slurries. The slurries were equal in concentration of
19
toothpaste to water, 1g/2mL, but the staturation onto filter discs may have differed. It is
possible the Colgate® discs leached their contents on the media surface because the sizes
of the zones were much larger than Crest®, Aquafresh®, and control groups. Since the
Colgate® zones were partly too large to be quantified, I would suggest placing one filter
disc on an inoculated agar plate rather than four discs per plate. I would also test a larger
sample size of 60 replicates for each of the toothpaste and control groups, which would
provide less variability and more confidence in the results.
Further studies could explore this experiment using revised methods to see if my
findings were upheld. One possibility for similar future experiments to test this
experiment’s findings would be to examine triclosan for its efficacy against S. mutans
under various conditions (Sullivan et al., 2003). Another topic to study would test if
there is a significant difference between fluoride and non-fluoride containing toothpastes
against S. mutans and other cariogenic (cavity-causing) bacteria using zone of inhibition.
Toothpaste is a major hygienic product that helps prevent the accumulation of oral
bacteria and reduces the chance of acquiring oral diseases. Brushing the teeth is an
important method that is commonly practiced because it is effective in its abrasive action
towards teeth. Since oral diseases are more likely to appear in people who do not brush
their teeth, toothpaste and other oral hygienic products are consistently used. Research
and statistics measure the impact of oral hygienic products and new methods of oral
hygiene will be tested to improve human oral health.
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Consent Form – Appendix 1
Approved: / / , IRB, Saint Martin's University
Consent to Act as a Subject in an Experimental Study
A Comparative Antibacterial Analysis of Three Marketed Toothpastes
Kevan Johanson
5001 College St. SE #H103 Lacey, WA 98503; (360)359-0215
Margaret Olney, Ph. D.
5300 Pacific Ave SE, Lacey, WA 98503 USA; (360)438-4327
Mary Jo Hartman, Ph. D.
5300 Pacific Ave SE, Lacey, WA 98503 USA; (360)438-4452
Source of Support: Department of Biology, Saint Martin's University
Description:
This study’s purpose is interpreting toothpaste efficacy on oral bacteria from
testing subjects’ mouths and examining zones of inhibition on Petri plates. In this study,
testing 60 subjects will provide a good sample size for testing toothpaste within a human
oral setting. Subjects will be required to be over the age of 18. Both genders and any
race/ethnicity will be accepted. The subjects will be orally swabbed on days one and
seven, and the duration will run for seven days. The subjects will brush their teeth twice
daily for two minutes for a week with their assigned toothpaste.
Risks and Benefits:
The only risk in this study is fluoride poisoning. Large quantities of toothpaste
should not be consumed.
Alternative Treatments:
There are no alternative treatments for this study.
New Information:
New information gained during the time the research is in progress and which is
relevant to participation will be provided.
Cost and Payments
There are no costs or payments associated with participating in this study.
______________
Subject’s Initials
21
Confidentiality:
I understand that any information about me obtained from this research, including
answers to questionnaires, history, laboratory data, findings on physical examination, or
audio or videotapes will be kept strictly confidential. Information that will carry personal
identifying material will be kept in locked files. I do understand that my research
records, just like hospital records may be subpoenaed by court order. It has been
explained to me that my identity will not be revealed in any description or publication of
this research. Therefore, I consent to such publication for scientific purposes.
Right to refuse or to end participation:
I understand that I am free to refuse to participate in this study or to end my
participation at any time and that my decision will not adversely affect my care at this
institution or cause a loss of benefits to which I might be otherwise entitled.
Voluntary Consent:
I certify that I have read the preceding or it has been read to me and that I
understand its contents. Any questions I have pertaining to the research have been and
will be answered by Kevan Johanson. Any questions I have concerning my rights as a
research subject will be answered by the Office of the Vice President for Academic
Affairs (360-438-4310). A copy of this consent form will be given to me. My signature
below means that I have freely agreed to participate in this experimental study.
___________
Date
____________________________
Subject Signature
__________________________
Witness
__________________________
************************************************************
Investigator’s Certification:
I, Kevan Johanson, certify that I have explained to the above individual the nature and
purpose, the potential benefits, and possible risks associated with participating in this
research study, have answered any questions that have been raised, and have witnessed
the above signature.
Signature of Investigator or Member of Research Staff:
Date: ________
______________________
Investigator/Research Staff
22
Acknowledgements
I would like to thank my participants in my study for their time and courage. I
also would like to thank Professor Olney and Professor Hartman because their input and
advice helped me in so many ways. I appreciate Cheryl Guglielmo for her assistance in
the lab. I would like to thank my colleagues in Senior Seminar for the peer-reviews and
input. Lastly, I thank the Biology Department of Saint Martin's University for funding of
my study.
23
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