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

Transcription

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 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

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 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 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) 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

11 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.

12 Resulting Number of Colonies Nicotine Effects on Yeast Survivorship Nicotine 0 0% 0.10% 1.00% 10% Nicotine Concentrations

13 Dunnett s Test T-Crit 3.1 Concentration T-value Interpretation.01% Nicotine Significant.1% Nicotine Significant 1% Nicotine Significant

14 Resulting Number of Colonies Ethyl Alcohol Effects on Yeast Survivorship Ethyl Alcohol % 1.00% 5.00% 10% Concentration of Ethyl Alcohol

15 Dunnett s Test T-Crit 3.1 Concentration T-value Interpretation.1%Ethyl Significant.5%Ethyl Significant 1%Ethyl Significant

16 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 )

17 Resulting Number of Colonies Nicotine and Ethyl Alcohol Effects on Yeast Survivorship 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)

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 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

20 Sources

21 Data Analysis Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column Column Column Column ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

22 Data Analysis (Cont.) Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column Column Column Column ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

23 Data Analysis ( Two Factor) Anova: Two-Factor With Replication SUMMARY 0.01%Nicotine 0.1%Nicotine 1%Nicotine Total 0.1%Ethyl Count Sum Average Variance %Ethyl Count Sum Average Variance %Ethyl Count Sum Average Variance

24 Two Factor (Cont.) Total Count Sum Average Variance ANOVA Source of Variation SS df MS F P-value F crit Sample E Columns E Interaction Within Total