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

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

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

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

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

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

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

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

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

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

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

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%.

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

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.

T-Values For 10% T-Crit: 2.18 Brand T-Value Listerine -1.1814576 Insignificant TopCare -1.181473 Insignificant Crest -2.98520 Significant Scope -2.87297 Significant

T-Values For 50% T-Crit: 2.18 Brand T-Values Listerine -2.21764 Significant TopCare -2.24736 Significant Crest -2.19573 Significant Scope -2.30857 Significant

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

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

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.

ANOVA Results for 10% Concentrations T-Critical=2.18

ANOVA Results for 50% Concentration T-Critical=2.18

Colony Count Table by Exposure Time