1. The Effects of Alcohol and Nicotine on Microbial Flora Jeff Van Kooten Grade 11 Pittsburgh Central Catholic High School

2. Microbial Flora The internal and external flora has eukaryotic fungi, protists, and bacteria Not much is known about the association between humans and their flora Effects can be mutualistic, parasitic, pathogenic, and commensal Provides nutritional and digestive benefits, secrete vitamins, stimulate antibody production, and protect against pathogenic microbes Substance intakes by humans might have unintended effects on these important functions of the flora

3. Ethyl Alcohol Often made through fermentation Fermentation is the process by which sugar is broken down by yeast into carbon dioxide and alcohol Used and abused by millions Can be used as an antiseptic

4. Nicotine derived from the tobacco plant considered a stimulant effects many areas in the body including the central nervous system and the endocrine (hormone) system Chewing tobacco Carcinogen Contains nicotine

5. Saccharomyces cerevisiae (Yeast) Used in many cell/biochemical investigations Easy to manipulate and rapidly grows As a eukaryote, it shares similar biochemistry, cell cycle, and genetics with more advanced organisms Utilized in this study as a human cell model and as a model of human Eukaryotic symbionts or pathogens

6. Purpose Test the effects of alcohol and nicotine individually and synergistically on Saccharomyces cerevisiae survivorship

7. Hypotheses Null: Nicotine and Ethyl Alcohol will not significantly effect yeast survivorship Alterative: Nicotine and Ethyl Alcohol will significantly reduce yeast survivorship individually Alternative: Nicotine and Ethyl Alcohol will interact synergistically to reduce yeast survivorship

8. Materials YEPD agar plates (YEPD media + 1.5% agar) YEPD media (1% yeast extract, 2% peptone, 2% glucose) Sterile dilution fluid [SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, 0.1mM CaCl2, 100mM NaCl) Sterile pipette tips Micropipettes Vortex Incubator Sidearm flask Spreading turntable Spreader bar Ethanol (Ethyl Alcohol) Sterile capped test tubes with Sterile distilled water. Hot plate Coffee filters Saccharomyces cerevisiae (Yeast) Flavorless chewing tobacco

9. Procedure 1.25 grams of chewing tobacco was mixed with 150mL of water 2. After thoroughly mixing the solution the tobacco leaves were drained out and the solution was filtered through coffee filters 3.The solution was then put on a hot plate and allowed to boil for 10 minutes (yielding a high [nicotine]) 4.Yeast was grown overnight in sterile YEPD media. 5.A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 6.The culture was placed in an incubator until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/mL. 7.The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 8.The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations of 0%, 0.1% 1.0% 5.0%, and 10% to a total of 9.9 mL. 9.0.1 mL of yeast was then added to the test tubes, yielding a final volume of 10 mL and a cell density of 10 3 cells/mL per tube.

10. Procedure (Continued) Concentrations0.0%.10% Nicotine1.0% (N)10.0% (N)1.0% Ethyl5.0% ( E )10.0% ( E ) Ethyl0mL.1mL.5mL1mL Nicotine0mL.01 mL.1mL1mL0mL Sterile Dilution Fluid9.9 mL9.89mL9.8mL8.9mL9.8mL9.4mL8.9mL Microbe.1mL Total Volume10mL Concentrations1.0% ( E ).10% (N)1.0% ( E ) 1.0% (N)1.0% ( E ) 10.0% (N)5.0% ( E ).10% (N)5.0% ( E ) 1.0% (N) Ethyl.1 ( E )mL 0.1 ( E )mL.5 ( E )mL Nicotine.01 (N)mL.1 (N)mL1 (N)mL.01 (N)mL.1(N) mL SDF9.79mL9.7mL8.8mL9.39mL9.3mL Microbe.1mL Total Volume10mL

11. Procedure (Continued) 7.The solutions were mixed by vortexing and allowed to sit at room temperature 8.After vortexing to evenly suspend cells, 0.1mL aliquots were removed from the tubes and spread on YEPD agar plates (6 plates per concentration) 9.The plates were then incubated at 30 degrees for 48 hours. 10.The resulting colonies were counted. Each colony is assumed to have arisen from one cell. Concentration5.0% ( E ) 10.0% (N)10.0% ( E ).10% (N)10.0% ( E ) 1.0% (N)10.0% (E) 10.0% (N) Ethyl.5 ( E )mL1 ( E )mL Nicotine1 (N) mL.01 (N)mL.1(N)mL1 (N)mL SDF8.4mL8.89mL8.8mL7.9mL Microbe.1mL Total Volume10mL

13. Dunnett’s Test ConcentrationT-valueInterpretation.01% Nicotine5.761243 Significant.1% Nicotine15.75422 Significant 1% Nicotine19.52709 Significant T-Crit 3.1

15. Dunnett’s Test T-Crit 3.1 ConcentrationT-valueInterpretation.1%Ethyl6.667737 Significant.5%Ethyl16.27352 Significant 1%Ethyl19.77707 Significant

16. Chart of concentrations 0%Nicotine.1% Nicotine1% Nicotine10% Nicotine 0%Ethyl0% (N) 0% (E ).1% N 0% (E )1% N 0% (E )10% N 0% (E ) 1%Ethyl0% (N) 1% (E ).1% (N) 1% (E )1% (N) 1% (E )10% (N) 1% (E ) 5%Ethyl0% (N) 5% ( E).1% (N) 5% (E )1% (N) 5%(E )10% (N) 5% (E ) 10%Ethyl0% (N) 10% (E ).1% (N) 10% (E )1% (N) 10% (E )10% (N) 10% (E )

18. Conclusion Increased concentrations of Nicotine appeared to significantly effect yeast survivorship Increased concentrations of Alcohol also appeared to significantly effect yeast survivorship Accept the first alternative hypothesis No evidence of synergic effects of nicotine and alcohol on yeast survivorship

19. Limitations and Extensions Limits Only yeast was used as a model Plating was not completely synchronized Pure nicotine was not used Only one kind of alcohol was used Extensions Use different concentrations of nicotine and ethyl alcohol Test carcinogenic effects Test on Escherichia coli populations

20. Sources http://www.encyclopedia.com/topic/tobacco.aspx http://en.wikipedia.org/wiki/Human_flora http://en.wikipedia.org/wiki/Gut_flora http://www.alcohol.vt.edu/Students/alcoholEffects/brainBody.htm http://www.alcohol.vt.edu/Students/alcoholEffects/brainBody.htm

21. Data Analysis Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column 1691815344.8 Column 26800133.333323.86667 Column 36634105.666718.66667 Column 4656894.6666717.06667 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups12617.3334205.778161.14093.56E-143.098391 Within Groups5222026.1 Total13139.3323

22. Data Analysis (Cont.) Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column 1691815344.8 Column 26805134.166748.16667 Column 36609101.524.7 Column 4653589.1666710.56667 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups15488.7935162.931161.0483.578E-143.098391 Within Groups641.16672032.05833 Total16129.9623

23. Data Analysis ( Two Factor) Anova: Two-Factor With Replication SUMMARY0.01%Nicotine0.1%Nicotine1%NicotineTotal 0.1%Ethyl Count66618 Sum7176365851938 Average119.510697.5107.6667 Variance16.319.623.5104.3529 0.5%Ethyl Count66618 Sum6354183751428 Average105.833369.6666762.579.33333 Variance18.9666735.066679.5399.5294 1%Ethyl Count66618 Sum5083792991186 Average84.6666763.1666749.8333365.88889 Variance15.06667885.366754.16667498.8105

24. Two Factor (Cont.) Total Count18 Sum186014331259 Average103.333379.6111169.94444 Variance232.2353652.6046455.9379 ANOVA Source of VariationSSdfMSFP-valueF crit Sample16373.4828186.74168.379012.28E-143.204317 Columns10626.0425313.01944.376512.27E-113.204317 Interaction1032.0744258.01852.1550760.0894862.578739 Within5387.66745119.7259 Total33419.2653