The Effects of Alcohol and Nicotine on Microbial Flora Jeff Van Kooten Grade 11 Pittsburgh Central Catholic High School
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
Ethyl Alcohol Fermentation is the process by which sugar is broken down by yeast into carbon dioxide and alcohol Used and abused by millions Alcohol is a depressant Alter the processes in the mind Can be used as an antiseptic
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
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
Purpose Test the effects of alcohol and nicotine individually and synergistically on Saccharomyces cerevisiae survivorship
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
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
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.
Procedure (Continued) Concentrations 0.0%.10% Nicotine 1.0% (N) 10.0% (N) 1.0% Ethyl 5.0% ( E ) 10.0% ( E ) Ethyl 0mL 0mL 0mL 0mL.1mL.5mL 1mL Nicotine 0mL.01 ml.1ml 1mL 0mL 0mL 0mL Sterile Dilution Fluid 9.9 ml 9.89mL 9.8mL 8.9mL 9.8mL 9.4mL 8.9mL Microbe.1mL.1mL.1mL.1mL.1mL.1mL.1mL Total Volume 10mL 10mL 10mL 10mL 10mL 10mL 10mL Concentrations 1.0% ( 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.1 ( E )ml 0.1 ( E )ml.5 ( E )ml.5 ( E )ml Nicotine.01 (N)mL.1 (N)mL 1 (N)mL.01 (N)mL.1(N) ml SDF 9.79mL 9.7mL 8.8mL 9.39mL 9.3mL Microbe.1mL.1mL.1mL.1mL.1mL Total Volume 10mL 10mL 10mL 10mL 10mL
Procedure (Continued) Concentration 5.0% ( E ) 10.0% (N) 10.0% ( E ).10% (N) 10.0% ( E ) 1.0% (N) 10.0% (E) 10.0% (N) Ethyl.5 ( E )ml 1 ( E )ml 1 ( E )ml 1( E )ml Nicotine 1 (N) ml.01 (N)mL.1(N)mL 1 (N)mL SDF 8.4mL 8.89mL 8.8mL 7.9mL Microbe.1mL.1mL.1mL.1mL Total Volume 10mL 10mL 10mL 10mL 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.
Resulting Number of Colonies 180 160 140 153 Nicotine Effects on Yeast Survivorship 134 120 100 80 60 40 20 101.5 89.166 Nicotine 0 0% 0.10% 1.00% 10% Nicotine Concentrations
Dunnett s Test T-Crit 3.1 Concentration T-value Interpretation.01% Nicotine 5.761243 Significant.1% Nicotine 15.75422 Significant 1% Nicotine 19.52709 Significant
Resulting Number of Colonies 180 160 153 Ethyl Alcohol Effects on Yeast Survivorship 140 133.33 120 100 105.66 94.66 80 Ethyl Alcohol 60 40 20 0 0.00% 1.00% 5.00% 10% Concentration of Ethyl Alcohol
Dunnett s Test T-Crit 3.1 Concentration T-value Interpretation.1%Ethyl 6.667737 Significant.5%Ethyl 16.27352 Significant 1%Ethyl 19.77707 Significant
Chart of concentrations 0.0%Nicotine 0.1% Nicotine 1.0% Nicotine 10% Nicotine 0.0%Ethyl 0.0% (N) 0% (E ) 0.1% N 0% (E ) 1.0% N 0% (E ) 10% N 0% (E ) 1.0%Ethyl 0.0% (N) 1% (E ) 0.1% (N) 1% (E ) 1.0% (N) 1% (E ) 10% (N) 1.0% (E ) 5.0%Ethyl 0.0% (N) 5% ( E) 0.1% (N) 5% (E ) 1.0% (N) 5%(E ) 10% (N) 5.0% (E ) 10%Ethyl 0.0% (N) 10% (E ) 0.1% (N) 10% (E ) 1.0% (N) 10% (E ) 10% (N) 10% (E )
Resulting Number of Colonies 180 160 153 Nicotine and Ethyl Alcohol Effects on Yeast Survivorship 140 120 100 80 60 119.5 105.833 84.66 106 69.66 62.5 97.5 81.5 49.83 40 20 0 Control.1%(N) 1%(E).1%(N).1%(N) 1%(N) 1%(N) 1%(N) 10%(N) 5%(E) 10%(E) 1% (E) 5% (E) 10% (E) 1% (E) Nicotine and Ethyl Concentrations 10%(N) 5% (E) 10%(N) 10%(E)
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
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 Limited amount of concentration Only survivorship accessed Cell health? Extensions Use different concentrations of nicotine and ethyl alcohol Test carcinogenic effects Test on Escherichia coli populations Growth curve
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.knowswhy.com/reasons-why-alcohol-is-bad-foryour-health/
Data Analysis Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 6 918 153 44.8 Column 2 6 800 133.3333 23.86667 Column 3 6 634 105.6667 18.66667 Column 4 6 568 94.66667 17.06667 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 12617.33 3 4205.778 161.1409 3.56E-14 3.098391 Within Groups 522 20 26.1 Total 13139.33 23
Data Analysis (Cont.) Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 6 918 153 44.8 Column 2 6 805 134.1667 48.16667 Column 3 6 609 101.5 24.7 Column 4 6 535 89.16667 10.56667 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 15488.79 3 5162.931 161.048 3.578E-14 3.098391 Within Groups 641.1667 20 32.05833 Total 16129.96 23
Data Analysis ( Two Factor) Anova: Two-Factor With Replication SUMMARY 0.01%Nicotine 0.1%Nicotine 1%Nicotine Total 0.1%Ethyl Count 6 6 6 18 Sum 717 636 585 1938 Average 119.5 106 97.5 107.6667 Variance 16.3 19.6 23.5 104.3529 0.5%Ethyl Count 6 6 6 18 Sum 635 418 375 1428 Average 105.8333 69.66667 62.5 79.33333 Variance 18.96667 35.06667 9.5 399.5294 1%Ethyl Count 6 6 6 18 Sum 508 379 299 1186 Average 84.66667 63.16667 49.83333 65.88889 Variance 15.06667 885.3667 54.16667 498.8105
Two Factor (Cont.) Total Count 18 18 18 Sum 1860 1433 1259 Average 103.3333 79.61111 69.94444 Variance 232.2353 652.6046 455.9379 ANOVA Source of Variation SS df MS F P-value F crit Sample 16373.48 2 8186.741 68.37901 2.28E-14 3.204317 Columns 10626.04 2 5313.019 44.37651 2.27E-11 3.204317 Interaction 1032.074 4 258.0185 2.155076 0.089486 2.578739 Within 5387.667 45 119.7259 Total 33419.26 53