1. The Effects of Chewing Tobacco on Microbial Flora Marco Augello Central Catholic HS Grade 10 Second Year in PJAS

2. Rationale Chewing tobacco is widespread in society, especially among young people Smokeless tobacco used regularly by 6.4% of high school students and 3% of adults

3. Chewing Tobacco Grizzly fine cut tobacco Small tobacco leaves consumed orally Causes tooth decay, gum disease, and other oral diseases Carcinogen – causes pancreatic, mouth, esophageal cancer Contains nicotine (highly addictive)

4. Staphylococcus epidermidis Bacteria found on healthy human skin Gram (+) After incubation in agar, forms 1/2 - 2 mm colonies Used as a model for microbial flora found in/on humans

5. Escherichia coli Large, diverse group of bacteria Bacteria found in intestines of most mammals Most strains are not pathogenic Gram (-) Used as a model for microbial flora found in the human body

6. Purpose To test the effects of chewing tobacco on Staph and E. coli populations.

7. Hypotheses Null Hypothesis: Chewing tobacco WILL NOT HAVE a significant effect on Staph and E. coli survivorship. Alternate Hypothesis: Chewing tobacco WILL HAVE a significant effect on Staph and E. coli survivorship.

8. Materials Escherichia coli (E. coli) Staphylococcus epidermidis (Staph) Grizzly fine cut tobacco Micro pipettes Sterile pipette tips Spreader bars Ethanol Bunsen burner Vortex Incubator Test tubes Filter paper Funnel Sterile filter Sterile Dilution Fluid (100mM KH2PO4, 100mM KH2PO4, 10mM MgSO4, 1mM NaCl) LB plates + media (1% tryptone, 0.5% yeast extract, 1% NaCl)

9. Liquid Pulse Procedure 1.Bacteria (E.coli and Staph) were grown overnight in sterile LB Media. 2.Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/mL. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 5. A 10% stock solution of chewing tobacco and sterile water was made. 6. The following components were added to sterile test tubes.

10. Table of Concentrations Liquid Pulse0%0.01%0.1%1% CT Solution (10%)0mL0.01mL0.1mL1mL SDF9mL8.99mL8.9mL8mL E. Coli/Staph1mL Total Volume10mL

11. Liquid Pulse Procedure cont’d 7.These solutions were then vortexed and allowed to sit at room temperature for 15 minutes. 8.After vortexing to allow even suspension, 100 μL aliquots were removed from the tubes and spread on LB-agar plates. 9.The plates were incubated at 37°C for 24 hours. 10.The resulting colonies were counted visually, each colony being assumed to have arisen from one cell.

12. Infusion Procedure 1.0.2ml of chewing tobacco concentrations (0%, 1%, and 10%) were spread on LB agar plates and incubated for 1 hour, allowing the bacteria to absorb the chemicals. 2. 100uL aliquots of bacterial suspensions from the original control tube were spread onto the infused plates. 3.The plates were incubated at 37 C for 24 hours. 4. The resulting colonies were counted visually, each colony being assumed to have arisen from one cell.

13. Chewing Tobacco Liquid Pulse Effects Chewing Tobacco Concentrations Average Colonies Counted Staph p-value=2.31x10 -5 E. coli p-value=0.000111

14. Dunnett’s Tests Stapht valuet criticalSignificance 0% to 0.01%2.3937570563.5NOT SIGNIFICANT 0% to 0.1%4.9833669623.5SIGNIFICANT 0% to 1%8.0734897073.5SIGNIFICANT E. colit valuet criticalSignificance 0% to 0.01%1.8426453863.5NOT SIGNIFICANT 0% to 0.1%5.2766663343.5SIGNIFICANT 0% to 1%6.3578880073.5SIGNIFICANT

15. Chewing Tobacco Infusion Effects Chewing Tobacco Concentrations Average Colonies Counted Staph p-value=8.39x10 -7 E. coli p-value=4.15x10 -7

16. Dunnett’s Tests Stapht valuet criticalSignificance 0% to 1%6.9322945934.25SIGNIFICANT 0% to 10%13.878128824.25SIGNIFICANT E. colit valuet criticalSignificance 0% to 1%7.6696161354.25SIGNIFICANT 0% to 10%15.056404784.25SIGNIFICANT

17. Conclusions The null hypothesis was REJECTED for all concentrations (liquid pulse and infusion) except for the liquid pulse 0.01% Experiment shows that chewing tobacco does have a significant and harmful effect on Staph and E. coli

18. Limitations Only survivorship tested Vitality of Staph/E. coli varied Spread plating may not have been synchronized One exposure period Limited replicates

19. Extensions More assessments Better plating synchronization Different brands/types of chewing tobacco More exposure times for liquid pulse

20. Resources http://kidshealth.org/teen/drug_alcohol/tobacco/sm okeless.html http://kidshealth.org/teen/drug_alcohol/tobacco/sm okeless.html http://betobaccofree.hhs.gov/health- effects/smokeless-health/ http://betobaccofree.hhs.gov/health- effects/smokeless-health/ http://www.cdc.gov/tobacco/data_statistics/fact_sh eets/youth_data/tobacco_use/index.htm#estimates http://www.cdc.gov/tobacco/data_statistics/fact_sh eets/youth_data/tobacco_use/index.htm#estimates http://www.ctri.wisc.edu/Smokers/smokeless_facts. htm http://www.ctri.wisc.edu/Smokers/smokeless_facts. htm