Brian Freyvogel
Central Catholic High School
Grade 9

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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

• 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

• 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

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

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

• Null 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

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

1.
E. coli was grown overnight in sterile LB Media.
2.
Samples of the overnight cultures were added to fresh media in a sterile sidearm flask.
3.
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.
4.
All four mouthwashes were diluted into 2 test tubes, each with concentrations of 50% and 10%.

Concentration 0% 10%
Bacteria
Mouthwash
Sterile Fluid
Total Fluid
0.1 mLs
0 mLs
9.9 mLs
10 mLs
0.1 mLs
1 mLs
8.9 mLs
10 mLs
50%
0.1 mLs
5 mL
4.9 mLs
10 mLs

Procedure cont.
5.
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.
6.
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
TopCare
Crest
Scope
-1.1814576 Insignificant
-1.181473 Insignificant
-2.98520 Significant
-2.87297 Significant

T-Values For 50% T-Crit: 2.18
Brand T-Values
Listerine -2.21764 Significant
TopCare
Crest
Scope
-2.24736 Significant
-2.19573
Significant
-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
Limitations
• 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
Jundishapur against four dental caries pathogens.
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