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

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

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

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

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

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

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

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.

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

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.

Concentrations and pH Values Chart 0% 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

Procedure (Continued) 6. 0.1 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.

Mouthwash Antimicrobial Activity at Different pH Levels Two-Factor P-Value<0.0000001 P-Value=0.299876 P-Value<0.0000001 P-Value<0.0000001 P-Value<0.0000001 7 pH P-Value<0.0000001

Dunnett's Test T-Crit=2.02979 Trial: T-Value Result 7 pH, 1% 16.8325 Significant 7 pH, 10% 17.8062 7 pH, 20% 16.9823 5 pH, 0% 18.0683 5 pH, 1% 15.5780 5 pH, 10% 17.8998 5 pH, 20% 17.7734

Dunnett's Test Continued T-Crit=2.02979 Trial: T-Value Result: 4 pH, 0% 16.9074 Significant 4 pH, 1% 15.5968 4 pH, 10% 18.4802 4 pH, 20% 34.5825 3 pH, 0% 17.3381 3 pH, 1% 17.6564 3 pH, 10% 33.2157 3 pH, 20% 34.8446

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%

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

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

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

Sources Bruckner, Monica Z. "Gram Staining." Microscopy. N.p., n.d. Web. 30 Nov. 2016. Bukhari, Muhammad. "Staphylococcus Epidermidis." Staphylococcus Epidermidis. N.p., 27 Sept. 2004. Web. 30 Nov. 2016. "COOL MINT LISTERINE® Antiseptic Mouthwash." LISTERINE®. N.p., n.d. Web. 30 Nov. 2016. Davis, Charles Patrick, PhD. "Staph Infection Pictures, Symptoms, Treatment & Contagious." EMedicineHealth. N.p., n.d. Web. 30 Nov. 2016. "The Definition of Acid." Dictionary.com. N.p., n.d. Web. 30 Nov. 2016. "Drugs@FDA Glossary of Terms." Drugs@FDA Glossary of Terms. N.p., n.d. Web. 30 Nov. 2016. "Hydrochloric Acid." Hydrochloric Acid - New World Encyclopedia. N.p., n.d. Web. 30 Nov. 2016. "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. 2016. "Learn More About Mouthrinses." Learn More About Mouthrinses. N.p., n.d. Web. 30 Nov. 2016.

Sources Continued "L-Menthol | C10H20O - PubChem." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 30 Nov. 2016. Modric, Jan. "Staphylococcus Epidermidis – Coagolase Negative Staphylococci." Healthhypecom. N.p., n.d. Web. 30 Nov. 2016. Osuna, Claudia. "What Is a Micropipette? - Pipette.com." Pipette.com. Claudia Osuna, 26 Mar. 2015. Web. 30 Nov. 2016. "PH Scale." PH Scale. N.p., n.d. Web. 30 Nov. 2016. "Staphylococcus Epidermidis Bacteria." Staphylococcus Epidermidis Bacteria. N.p., n.d. Web. 30 Nov. 2016. "THYMOL | C10H14O - PubChem." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 30 Nov. 2016. "Tips for Limiting Acidic Foods." Healthline. N.p., n.d. Web. 30 Nov. 2016. "What Does PH Stand For?" About.com Education. N.p., 30 Nov. 2015. Web. 30 Nov. 2016. Mitchell, Jennifer. "How Acidic Drinks Affect Teeth." How Acidic Drinks Affect Teeth. N.p., 04 Jan. 2016. Web. 23 Jan. 2017.

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

Two-Factor Anova

7 pH ANOVA

0% ANOVA

1% ANOVA

10% ANOVA

20% ANOVA