1. Acidic Effects of Mouthwash on Bacterial Survivorship
Jack Zagrocki
Central Catholic High School
Grade 9

2. Problem
Does an increase in the acidity of the environment affect the efficiency of mouthwash on Staphylococcus Epidermidis survivorship?

3. Purpose Increase in consumption of acidic foods
Include fruits, carbonated beverages, dairy products, meats, and a multitude of processed goods
How does this affect oral hygienic products?
Does it make them stronger?
Weaker?
Test how these conditions affect mouthwash

4. Staphylococcus Epidermidis
Gram-positive
Coagulate-negative
Anaerobic
Prokaryotic
Commonly found on human flora (skin)
Can infect human body
Thrives in a neutral environment
Tolerates pH levels of 5.5-8

5. Listerine Mouthwash
Anti-bacterial Mouthwash designed to kill germs that cause bad breath, plaque, and gum disease
Uses active ingredients and other elements to keep the mouth healthy
Other elements include antimicrobial agents, fluoride, astringent salts, odor neutralizer

6. Listerine Ingredients
Active
Inactive
0.092% Eucalyptol
O.042% Menthol
0.060% Methyl Salicylate
0.046% Thymol
Water
Alcohol (21.6%)
Sorbitol solution
Various flavorings
Poloxamer 407
Benzoic acid
Sodium saccharin
Sodium benzoate
FD&C Green No. 3

7. Hydrochloric Acid Abbreviated as HCl
Water based solution of hydrogen chloride gas
Strong, corrosive, dangerous

8. Hypothesis Alternative Null
The acid and mouthwash will have no individual effects on bacterial survivorship and there will be no interaction between the acid and the mouthwash.
The acid and the mouthwash will produce individual negative effects on the bacterial survivorship and there will be interaction between the two variables.

9. Materials Staphylococcus Epidermidis LB agar plates
LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride
Hydrochloric Acid
Listerine Cool Mint Mouthwash
Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
Vortex
Sharpie marker
Incubator
Sterile Filter
Spreader Bars
Sterile test tubes
P1000 micro-pipette
P100 micro-pipette
Sterile pipette tips
Bunsen burner
Klett spectrophotometer
Sidearm flask
Test tube rack
Ethanol

10. Procedure
Staphylococcus Epidermidis was grown overnight in sterile LB media
A sample of the overnight culture was 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 10^8 cell/mL.
The culture was diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL.
Various mouthwash concentrations of 0%, 1%, 10% and 20%, along with pH values of 7, 5, 4, and 3 were created in test tubes containing SDF resulting in 9.9mL of fluid per tube. The various pH levels were created using HCl with a 0.1 molar concentration.

11. Concentrations and pH Values Chart0% 1%
10%
20%
pH of 7
9.9ml sf
9.8ml sf
8.9ml sf
7.9ml sf
0ml HCl
0ml mw
0.1ml mw
1ml mw
2ml mw
pH of 5
9.84ml sf
9.74ml sf
8.784ml sf
9.784ml sf
0.06ml HCl
pH of 4
9.82ml sf
9.72ml sf
8.82ml sf
7.82ml sf
0.08ml HCl
pH of 3
9.7ml sf
8.8ml sf
7.8ml sf
0.1ml HCl

12. Procedure (Continued)
ml of cell culture was then added to the test tubes, yielding a final volume of 10 mL.
7. The solutions were mixed by vortexing and allowed to sit at room temperature for 10 minutes.
8. After vortexing to evenly suspend the cells, 0.1 ml was removed from each tube and spread onto the LB plates, resulting in 5 plates per tube.
9. The plates were incubated at 37°C for 24 hours
10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

13. Mouthwash Antimicrobial Activity at Different pH Levels
Two-Factor P-Value<
P-Value=
P-Value<
P-Value<
P-Value<
7 pH P-Value<

14. Dunnett's Test T-Crit=2.02979 Trial: T-Value Result 7 pH, 1% 16.8325
Significant
7 pH, 10%
7 pH, 20%
5 pH, 0%
5 pH, 1%
5 pH, 10%
5 pH, 20%

15. Dunnett's Test Continued
T-Crit=
Trial:
T-Value
Result:
4 pH, 0%
Significant
4 pH, 1%
4 pH, 10%
4 pH, 20%
3 pH, 0%
3 pH, 1%
3 pH, 10%
3 pH, 20%

16. Conclusions Null hypothesis is rejected
The mouthwash and acid had a significant negative effect on bacterial survivorship.
Mouthwash and Acid also interacted to have a significant negative effect on bacterial survivorship.
Both variables were powerful at lower concentrations and were very deadly when combined at high concentrations
The acid had significant effect on the mouthwash at all concentrations except 1%

17. Conclusion Continued
Possibly due to high alcohol concentration within the mouthwash and the pH value being outside of the bacteria's tolerance range
Alcohol is a well-known anti-septic and all of the pH values were lower than 5.5, the lowest value studies show the bacteria are not affected at
While the bacteria were not able to survive in this situation, acid has a negative effect on oral health
Breaks down the enamel, weakens the overall structure of teeth, makes the tooth more sensitive, and causes many painful diseases and conditions

18. Limitations Only one type of bacteria was tested
Only one exposure time
Only one type of mouthwash used
Instruments used could have been slightly contaminated
Plating, vortexing, pipetting, and exposure times could have been slightly unsynchronized
Study does not account of how other factors affected bacteria survivorship

19. Extensions Use different types of bacteria
Use other brands and flavors of mouthwash
Use different mouthwash concentrations and pH values
Use different exposure times
More additional trials per group
More control in human elements of experiment

20. Sources
Bruckner, Monica Z. "Gram Staining." Microscopy. N.p., n.d. Web. 30 Nov
Bukhari, Muhammad. "Staphylococcus Epidermidis." Staphylococcus Epidermidis. N.p., 27 Sept Web. 30 Nov
"COOL MINT LISTERINE® Antiseptic Mouthwash." LISTERINE®. N.p., n.d. Web. 30 Nov
Davis, Charles Patrick, PhD. "Staph Infection Pictures, Symptoms, Treatment & Contagious." EMedicineHealth. N.p., n.d. Web. 30 Nov
"The Definition of Acid." Dictionary.com. N.p., n.d. Web. 30 Nov
Glossary of Terms." Glossary of Terms. N.p., n.d. Web. 30 Nov
"Hydrochloric Acid." Hydrochloric Acid - New World Encyclopedia. N.p., n.d. Web. 30 Nov
"Influence of the PH-value on the Growth of Staphylococcus Epidermidis, Staphylococcus Aureus and Propionibacterium Acnes in Continuous Culture." Zentralblatt Fur Hygiene Und Umweltmedizin = International Journal of Hygiene and Environmental Medicine. U.S. National Library of Medicine, n.d. Web. 30 Nov
"Learn More About Mouthrinses." Learn More About Mouthrinses. N.p., n.d. Web. 30 Nov

21. Sources Continued
"L-Menthol | C10H20O - PubChem." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 30 Nov
Modric, Jan. "Staphylococcus Epidermidis – Coagolase Negative Staphylococci." Healthhypecom. N.p., n.d. Web. 30 Nov
Osuna, Claudia. "What Is a Micropipette? - Pipette.com." Pipette.com. Claudia Osuna, 26 Mar Web. 30 Nov
"PH Scale." PH Scale. N.p., n.d. Web. 30 Nov
"Staphylococcus Epidermidis Bacteria." Staphylococcus Epidermidis Bacteria. N.p., n.d. Web. 30 Nov
"THYMOL | C10H14O - PubChem." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 30 Nov
"Tips for Limiting Acidic Foods." Healthline. N.p., n.d. Web. 30 Nov
"What Does PH Stand For?" About.com Education. N.p., 30 Nov Web. 30 Nov
Mitchell, Jennifer. "How Acidic Drinks Affect Teeth." How Acidic Drinks Affect Teeth. N.p., Jan Web. 23 Jan

22. Full Data Table 0% 1%
10%
20% 7
389
210
158
211
327
226
185
188
403
166
200
180
373
181
173
191
369
179
194
184 5
149
139
242
189
186
171
182
156
221
165
167 4
193
144 1
187
195
174 2
225
207
228
169
197
190 6 3 14 17
178 24
196 13
175 19

23. Two-Factor Anova

24. 7 pH ANOVA

25. 0% ANOVA

26. 1% ANOVA

27. 10% ANOVA

28. 20% ANOVA