1. Antimicrobial Abilities of Top-Selling Mouthwashes
Brian Freyvogel
Central Catholic High School
Grade 9

2. Crest Pro—Health Multi-Protection Mouthwash
Active Ingredient:
Cetylpyridinium chloride 0.07%
Alcohol FREE

3. Scope Outlast Mouthwash
Active Ingredients:
Cetylpyridinium chloride 0.07%
Alcohol based

4. Listerine Cool Mint Antiseptic Mouthwash
Active Ingredients:
Eucalyptol 0.092%
Menthol 0.042%
Methyl salicylate 0.060%
Thymol 0.064%
Claims to kill 99.9% of bacteria
Alcohol based

5. TopCare Antiseptic Mouth Rinse
Active Ingredients:
Eucalyptol 0.092%
Menthol 0.042%
Methyl salicylate 0.060%
Thymol 0.064%
Alcohol based

6. Escherichia coli (E. coli)
Large and diverse group of gram(-) bacteria
Free living, symbionts, or pathogens
Lives in the intestinal tract of many
mammals
Serves as a common prokaryotic
cell model
Commonly used as non-target
bacteria for testing of potential
antimicrobial abilities

7. Previous Studies
According to Listerine’s official site, they did an experiment in which they tested their product and it killed 99.9% of bacteria.
Research was also done by Shelby Dental Care, and this found Crest to be a more advantageous mouthwash than Listerine

8. Problem
Is there a significant difference in the antimicrobial abilities of these mouthwashes?

9. Purpose
To determine if there is significant variation in the effects of various mouthwashes on E. coli survivorship

10. Hypothesis Null hypothesis: Alternate Hypothesis:
There will not be significant variation among the effects of the mouthwashes on the survivorship of E. coli
Alternate Hypothesis:
There will be significant variation among the effects of the mouthwashes on the survivorship of E. coli

11. Materials Listerine Cool Mint Antiseptic mouthwash
Open flame and ethanol (for sterilization)
Scope Outlast mouthwash
Various sized pipettes
Sterile fluid
Crest Pro-Health Multi-Protection mouthwash
Test tubes
Vortex
Incubator
TopCare Antiseptic Mouth Rinse
Klett Spectrophotometer
Sidearm flask
E. coli
Spreader bars

12. Procedure E. coli was grown overnight in sterile LB Media.
Samples of the overnight cultures were added to fresh media in a sterile sidearm flask.
The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10⁸ cells/mL.
All four mouthwashes were diluted into 2 test tubes, each with concentrations of 50% and 10%.

13. Procedure Cont. Concentration 0% 10% 50% Bacteria 0.1 mLs Mouthwash
Sterile Fluid
9.9 mLs
8.9 mLs
4.9 mLs
Total Fluid
10 mLs

14. Procedure cont.
0.1 mL of the bacteria was added to each tube, creating a 10^3 cells/mL cell concentration. The tubes were vortexed. Then 0.1 mL was pipetted to the Agar plate after exposure times of 30 seconds, 1 minute, and 5 minutes were reached. The plates were spread and placed in an incubator for 24 hours at 37°C.
The resulting cell colonies were counted. All colonies were assumed to have risen from one cell.

16. T-Values For 10% T-Crit: 2.18 Brand T-Value Listerine
Insignificant
TopCare
Insignificant
Crest
Significant
Scope
Significant

17. T-Values For 50% T-Crit: 2.18 Brand T-Values Listerine
Significant
TopCare
Significant
Crest
Significant
Scope
Significant

18. Conclusions REJECT NULL for:
Crest and Scope 10% concentration tests
All 50% concentration tests
T-Value of Scope and Crest was greater than the T-crit.
T-Value of Listerine and TopCare was lower than the T-crit.
Scope and Crest more effective, greater antimicrobial ability

19. Limitations and Future Studies
Only two concentrations were used
Only three exposure times were used
Only a single, non-target bacteria was used
Only liquid pulse was tested
The mouthwashes were not tested at full strength
Future Studies
Use more mouthwash brands
Use higher concentrations
Test cost-efficiency
Agar infusion tests
Test more generic mouthwash brands
Include test of exposure time
Test Streptococcus Mutans

20. References
Anayanwu, O. C., K.K. Baugh, S.B. Bennett J. M. Johnson, R. L. Madlock, N. E. Pollard, and J. O. Chikwem. “Comparison of the Antimicrobial Effectiveness of Alcohol- Containing and Non-alcohol-containing Mouthwashes.” Diss. Lincoln U, n.d. Abstract.. (n.d.): n. pag. Web.
Aneja KR, Joshi R, Sharma C. The anti microbial potential of ten most often used mouthwashes against four dental caries pathogens. Jundishapur J Microbiol. 2010; 3(1): 15-27
Masadeh, Majed M., Shadi F. Gharaibeh, Karem H. Alzoubi, Sayer I. Al-Azzam, and Wasfi M. Obeidat. “Antimicrobial Activity of Common Mouthwash Solutions on Multidrug- Resistance Bacterial Biofilms.” N.p., n.d. Web.

21. ANOVA Results for 10% Concentrations
T-Critical=2.18

22. ANOVA Results for 50% Concentration
T-Critical=2.18

23. Colony Count Table by Exposure Time