Download Evaluation and Comparison of Bacterial Growth in the

Do Crest Whitestrips® reduce oral bacterial levels in vivo and in vitro?
Bridgette Losey
Senior Seminar
May 10, 2007
Final Draft
Saint Martin’s University
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Table of Contents
Abstract…………………………………………………………………………………1
Introduction……………………………………………………………………..…..….2-7
Methods...........................................................................................................................7-9
Participant Questionnaire……………………………………………………………….10
Results………………………………………………………………………………….11-13
Discussion……………………………………………………………………………….
Acknowledgements………………………………………………………………………
Literature Cited..................................................................................................................
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Abstract:
This study was undertaken to examine whether Crest Whitestrips® reduced
concentrations of bacteria present in the human oral cavity. Fifteen participants used Crest
Whitestrips® and a separate 15 did not use Crest Whitestrips® for 7 days. The participants’
mouths were swabbed on day 1 and day 7 of the study. The swabs were incubated in nutrient
broth and the broths bacterial absorbance was measured using a spectrophotometer. Also, a zone
of inhibition test was used to determine if Crest Whitestrips® inhibited growth in a laboratory
setting. Results were compared with a t-test, and these results showed no difference in the levels
of bacteria in the human oral cavity whether the participants used or did not use Crest
Whitestrips®. The Crest Whitestrips® did inhibit bacterial growth in the laboratory setting.
Introduction:
A recent trend in oral and cosmetic care is the use of over-the-counter teeth bleaching
products that contain carbamide peroxide and/or hydrogen peroxide. There are brand name
products that claim to whiten teeth in less than seven days, such as: Crest Whitestrips®.
However, there is little understanding of what effect the whitening products have on oral health
and bacterial growth. It is important to understand whether at-home bleaching products help,
hinder, or have no effect on bacterial growth in the human oral cavity. If the products reduce the
amount of bacteria in the oral cavity, this could help the companies market their products and
would be useful information for consumers. However, if the products increased the amount of
bacteria in the oral cavity, this would be information that the people using the products should
know.
Kihn et al., 2000 conducted a clinical evaluation testing the effectiveness of peroxide in
at-home bleaching products. This study tested the difference between 10% and 15%
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concentrations of carbamide peroxide treatments on tooth whitening. Kihn et al. (2000) used 57
human subjects in a double-blind study. They determined that after one week, there was no
change in sensitivity or color. This study relates directly to the methods that were utilized in my
study. Similar to my study, if the experiment was going to be conducted again, it would be
useful to have a larger sample group and have the control group think that they were actually
using Crest Whitestrips®.
Another study tested the effectiveness of a brand of whitening system. The Gold Tooth
Whitening System™ (Barnes et al., 1998) was tested on 60 participants over a two week period.
This double-blind study found that a placebo whitened teeth by less than 1 shade, but that the
Gold Tooth Whitening System™ whitened teeth almost 7 shades. Teeth sensitivity was also
compared and ranged from not sensitive to extremely sensitive. Teeth sensitivity was monitored
by a pre-experiment survey asking the subjects if they have experienced tooth sensitivity after
using an at-home bleaching product in the past. The Barnes et al. (1998) stated the people who
had experienced teeth sensitivity in the past were prone to experiencing sensitivity again. The
Barnes et al. (1998) study directly related to my study, because I conduct ed a pre-experimental
survey in order to determine which participants would be good candidates to use Crest
Whitestrips®.
During the first week of Heymann et al.’s (1998) study the participants used a placebo gel
and then used an experimental gel for the second week. The study used the Vita shade guide to
compare the whitening ability of the various bleaching gels. Side effects including tooth
hypersensitivity and gingival irritation were assessed by participants keeping a daily diary of
how their teeth and gums felt. Heyman et al. (1998) found there was no change of shade after 5
days of gel use. Also, there were no changes in plaque or gingival irritation, and only mild tooth
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sensitivity occurred after using both whitening systems. This study explained how important it is
to look at all the variables that can affect the study. The participants of this study were evaluated
prior to participating in this study. Oral hygiene and daily regimes were taken into consideration
when evaluating potential participants. I also wish to conduct a pre-experimental survey to
determine the level of oral hygiene care and maintenance of potential subjects (Appendix 1).
Sagel et al. (2000) evaluated the effectiveness of trays, strips, and paint-on bleach. The
study used participants ranging from 21-65 years of age, and each participant was given a
different product to use for a 2 week period. All products contained the same amount of
peroxide, but differed in types of application. The participant surveys concluded that Crest
Whitestrips® were the easiest to use. The Vita Shade guide determined the Crest Whitestrips®
achieved the greatest shade change. Although there are numerous teeth whitening products
available on the market from Sagel et al., (2000) I determined I would use Crest Whitestrips® in
my study.
A 2 year study was conducted by Swift et al., (1999). This study had a sample group of
91 patients use whitening gel (10% carbamide peroxide) on their maxillary teeth, the upper row
of teeth, for 2 weeks, once daily. The patients were tested after the two weeks and an average
change of 7 shades was found. The patients were then recalled after two years to determine
whether their teeth had returned back to the original shades. Teeth that were treated remained
whiter than teeth that were not treated. Swift et al., (1999) used the Vita shade guide and
recorded the results before treatment, after two weeks of treatment and again two years later.
From this, they determined the shade changes and statistically analyzed them using a multiple
comparisons test. I intend
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My hypothesis was that Crest Whitestrips® would reduce the concentration of bacteria
present in the human mouth. Hydrogen peroxide (H2O2) is commercially marketed as a
disinfectant and bleaching agent. The hydrogen peroxide and carbamide peroxide in the Crest
Whitestrips® should result in lowering the amount of bacteria found in the oral cavity. I also
believe that the same situation of reducing oral bacteria after the use of Crest Whitestrips® can
be replicated in the lab. The lab setting will possibly be more effective because of the variables
that occur when using human participants.
Methods:
This study began by swabbing the mouths of 15 participants who would be using Crest
Whitestrips®, labeled group A. These participants were swabbed once before using the strips
and once after, with 7 days separating each swabbing. A second group of 15 participants,
labeled group B, were swabbed before and after the study. Group B participants had not used
any teeth whitening treatment within 1 month of the study and therefore were the control group.
When initially participating in the study the subjects did not know whether they would or would
not be using the Crest Whitestrips®. Group A participants used Crest Whitestrips® and were
instructed to brush their teeth for 2 minutes twice daily with a provided Crest® medium bristle
toothbrush and Crest® toothpaste for the 7 day duration of the study (Heymann et al., 1998).
The toothbrush and toothpaste were provided by West Side Dental Clinic located in Olympia,
Washington. Providing the participants with brushing materials was done in order to avoid
variables that could be attributed to individuals using different toothbrushes and toothpastes.
Between the first brushing and the initial swab, which was on average 4 hours, the participants
were instructed to refrain from eating any food or drinking any beverages except water.
A swab of each participant’s mouth was taken in a sterile method. The swab was taken
from the maxillary anterior teeth and mandibular posterior molars on both sides of the mouth
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(Heymann et al., 1998). The swab was placed in a test tube with Fisher® nutrient broth and
incubated at 37o C for an initial 24 hours and then for an additional 24 hours (Ahn et al., 2006).
After the initial 24 hours, the optical density of the bacterial growth was measured using a
spectrophotometer. This was measured again after a total of 48 hours had passed since the
participants had been swabbed. This specific temperature was used because it is the average
human body temperature, and the 24 and 48 hours was derived from the study by Ahn et al.,
(2006). Each test tube of nutrient broth contained 10mL of broth and had been autoclaved in a
Tuttnauer™ 3870E autoclave at 121 o C at 15psi.
The control group, group B, had not used at-home bleaching treatments within the last
month. However, variables that are seen in the human subject, such as diet, oral hygiene and the
use of the strip varied. These variables were only partially eliminated through the use of the
survey before the subjects participated in the study. Strict limitations on diet were not enforced
for this study because of Institutional Review Board constraints.
Subject Recruitment:
The 30 participants ranged from 18-32 years of age. A survey, (Appendix 1), was
conducted to ensure the participants had a professional cleaning within the last year and brushed
at least twice daily. I instructed the Crest Whitestrips® users, group A, to use the strip for 7
days, 30 minutes each day, after they flossed and brushed their teeth. Group A participants used
the toothbrushes and toothpastes that was provided. After using the strips, the participants were
instructed to rinse their mouth with tap water (Heymann et al., 1998). Seven days after using the
Crest Whitestrips® the same participants from group A were instructed to return to the lab and
the same swabbing procedure was conducted as on day 1 of the experiment. The participant’s
mouth was swabbed again using a sterile technique. After incubating the nutrient broth tubes
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with the samples, the oral bacterial concentrations were tested using a spectrophotometer, or
commonly called a spec. A spec is a device that measures the quantity of substances and their
ability to absorb light at various wavelengths. Before any samples were measured the spec was
zeroed using a cuvette with sterile nutrient broth. The wavelength that the spec was set to was
686nm. The model of the spec that was used for this experiment was Spectronic 20D+
manufactured by Spectronic Instruments™.
Methods II:
In the laboratory setting, 1 set of Petri agar plates, that contained approximately 15ml of
Fisher® nutrient agar were prepared. The agar was autoclaved in a Tuttnauer™ 3870E autoclave
at 121 oC at 15psi and then transferred to the Petri plates. The nutrient broth from mouth bacteria
was pipetted onto the Petri plates. The bacterial lawns were cultivated over a 48 hour period of
time. A total of 30 plates were divided in half, side A contained the Crest Whitestrip®, and side
B contained a piece of plastic with the same consistency of the Crest Whitestrip®. These 30
Petri agar plates had a 1x1 inch Crest Whitestrip® placed on the bacterial lawn, using sterile
forceps, and left for 30 minutes on side A. On side B a 1x1 inch piece of plastic was placed on
the bacterial lawn and left for 30 minutes. The strips were removed and new strips were used for
the duration of 7 days in a row on the same 30 plates. These procedures were conducted to
simulate what the participants were doing with the strips at home. The zone of inhibition test
was utilized to determine if the Crest Whitestrips® inhibited the growth of oral bacteria in a
laboratory setting. The zone of inhibition test was measured by diameter in millimeters of where
the bacteria did not grow when the strip was placed on the plate.
Statistical Analysis:
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A paired t-test was completed via Microsoft Excel (2003) in order to determine if there
was a difference between the bacterial levels in oral cavities of Crest Whitestrips® users before
and after the study was completed. The absorbance of bacteria between the control group and
the pre-swab of the Crest Whitestrip® users were compared using a t-test. I also conducted a ttest on the zone of inhibition plates.
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Participant Questionnaire
You are invited to participate in a student directed research project for Biology Senior
Seminar. This study will determine the change in oral bacterial concentrations that Crest
Whitestrips® has on 25 Saint Martin’s University students. To participate in this study
you will only need to fill out the attached questionnaire. There will be complete privacy
of the information you supply, because your name will never be used or associated with
the project. You are free to choose to participate or not participate, and you may stop
your participation at any time. Whether you participate or not will have no detrimental
effect on your relationship with the University. By completing this questionnaire, you
are giving your consent to participate in the project, and you are certifying that you are
over 18 years old.
Dental Questionnaire:
Please circle your answer
1. Have you used a tooth whitening product in the last month?
Yes
No
2. Have you ever experienced tooth sensitivity while using a tooth whitening product?
Yes
No
3. Do you brush your teeth at least twice daily?
Yes
No
4. How many times a day do you brush exactly?
0
1
2
3
4
5
5. Do you floss at least once daily?
Yes
No
6. When is the last time you have had your teeth professionally cleaned?
Within the last 6 months
Within the last year
Longer than a year
7. Do you have: caps, crowns, veneers, fillings, dentures, or braces?
Yes
No
8. Would you have twenty minutes in the evening every night for seven days to use Crest
Whitestrips®?
Yes
No
Results
Statistical analyses were run in Microsoft Excel® (2003). A t-test between the control
group (group B) and the pre-swab of Crest Whitestrips® (group A) showed that there was
no significant difference between the two groups (t=2.056; d.f.=26; p=0.35). The paired
t-test showed that there was no significant difference between group A, before using
Crest Whitestrips® and after using Crest Whitestrips® (t=2.16; d.f.=13; p=0.28).
Figure 1 shows the absorbency of bacteria at 24 hours.
Figure 1.
0.18
0.16
Absorbance
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
Control
Before C.W.
After C.W.
Figure 1. The absorbency of bacteria after 24 hours of incubation at 37o C. The standard error bars
represents one standard error. C.W. represents Crest Whitestrips®. The three bars represent two different
groups of people, before and after C.W. are one set of people.
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Figure 2 shows the absorbency at 48 hours.
Figure 2.
1.2
Absorbance
1
0.8
0.6
0.4
0.2
0
Control
Before C.W.
After C.W.
Figure 2. The absorbency of bacteria after 48 hours of incubation at 37o C. The standard error bar
represents one standard error. C.W. represents Crest Whitestrips®. The Y axis scale is different than
Figure 1. because the bacteria grew substantially between 24 and 48 hours. The three bars represent two
different groups of people, before and after C.W. are one set of people.
A t-test was also completed for the zone of inhibition portion of the experiment.
The t-test showed that there was no significant difference in inhibiting bacteria growth
between the control and the Crest Whitestrip® (t=2.00; d.f.=56; p=0.55).
Figure 3.
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Discussion:
This study did not support my hypothesis that using Crest Whitestrips® would
inhibit the growth of bacteria in the oral cavity. According to the data analysis, there
were no significant differences in bacterial concentrations between using and not using
Crest Whitestrips® (t=2.16; d.f.=13; p=0.28). There are several possibilities as to why
these results were obtained.
A survey (Appendix 1) was conducted before any data was collected to determine
the oral hygiene of each individual participating in the study. I only included people in
group A (the Whitestrip® using group) if they had a professional dental cleaning within
the last year and brushed their teeth at least twice daily. This was to ensure that my
participants had good oral hygiene. However, this could have also potentially altered my
results because people who are concerned about teeth whitening most likely already have
good oral hygiene habits. Therefore, a considerable reduction in oral bacteria in these
participants would not have been likely to be seen.
The participants that were included in my study were volunteering their time. I
couldn’t restrict sugar intake and put limitations on their diet because this would be an
unreasonable request to require of my participants. It is possible that if I restricted their
diet I would have no participants in my study. If my subjects consumed an increased
amount of sugar one days this could increase the oral bacteria levels that would be seen
and therefore in crease the absorbance readings (Newbrun, 1982.)
If the study were going to be conducted again, there are some modifications that I
would recommend and a more direct question that should be posed. A large sample size
was not considered for this study because of cost limitations, but I believe a larger sample
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size would be beneficial in order to determine whether it was just the participants that I
had in this study or if Crest Whitestrips® really don’t have any antibacterial properties. I
would also recommend using tryptic soy agar and nutrient broth because Streptococcus
mutans prefer this medium and S. mutans is a predominant oral bacterium (Park et. al.,
2004). If the zone of inhibition portion of the lab was going to be completed again I
would recommend taking the gel from the Whitestrips® and directly applying it to the
agar. This is because the strips are very flexible and the flexibility did not allow for good
contact with the agar. For the zone of inhibition, the control worked better than the Crest
Whitestrips®. I believe this is because the control plastic had better contact to the agar
and allowed less oxygen to flow to the area and therefore did not allow as much bacteria
to grow.
Over-the-counter teeth bleaching continues to be a popular trend in oral and
cosmetic care. However, there is still little understanding as to what effect whitening
products have on oral health and bacterial growth. It is important to understand if athome bleaching products help, hinder, or have no effect on bacterial growth in the human
oral cavity because these products are so popular and there is use is wide-spread. This is
why more research needs to be done on the antibacterial effects of at-home bleaching
products.
Acknowledgments:
I would first like to thank the participants of this study for volunteering their time
and my peers for offering their advice. I would like to give Dr. Hartman and Dr. Olney a
special thank you for giving their opinions, advice and knowledge throughout the entire
duration of this project. However, most importantly the experiment would have not been
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completed if it wasn’t for Cheryl Guglielmo who gave me her patience and expertise in
the laboratory. Thank you to everyone who supported me throughout this process.
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Ahn, H.J., Song, K.B., Lee, Y.E., Lee, J.T., Cho, S.A., Kim, KH. 2006. Surface change of
dental amalgam after treatment with 10% carbamide peroxide. Dental Materials
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Barnes, D.M., Kihn, P.W., Romberg, E., George, D., DePaola, L., Medina, E. 1998.
Clinical evaluation of a new 10% carbamide peroxide tooth-whitening agent.
Compendium Continuing Education in Dentistry. 19: 968-72.
Heymann, H.O., Swift, E.J., Bayne, S.C., May, K.N., Wilder, A.D., Mann, G.B.,
Peterson, C.A. 1998. Clinical evaluation of two carbamide peroxide toothwhitening agents. Compendium Continuing Education in Dentistry. 4: 359-69.
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Peña, A.D., Cabrita, B.O. 2006. Comparison of the clinical efficacy and safety of
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with a novel hydrogen peroxide strip system. Compendium Continuing Education
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Swift, E.J., May, K.N., Wilder, A.D., Heymann, H.O., Bayne, S.C. 1999. Two-year
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