1. Cigarette Leachate Effects on Microbial Survivorship By Jack Devine

2. Purpose to investigate the effects of tobacco product fragments on yeast cells bacterial cells, and tobacco to determine its toxicity

3. Null/Alternative Hypothesis Null: Tobacco product fragments will not significantly alter microbial survivorship Alternative: Tobacco product fragments will significantly alter microbial survivorship

4. Environmental Dangers of Tobacco Fire hazard EPA calls second hand smoke “an environmental toxin equivalent to asbestos” Trees needed for paper –One tree for every 300 cigarettes Discarded cigarette butts –Wash into rivers –Decompose into soil

5. Yeast Background Used in baking and alcohol fermentation Most studied eukaryotic cell –Similar structure to human cells Saccharomyces cervevisiae yeast cells

6. E. coli Background Used in Long-Term Evolution Project Most studied prokaryotic cell –Similar structure to human cells E. coli cells

7. Dunnett’s Test If P-Value>.05, then there is significant variance If the t-value>3.10, then there is significant variance

8. Materials YEPD Plates LB Plates YEPD Media (0.5% yeast extract, 2% peptone, 2% glucose) LB Media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Klett Spectrophotometer Sterile sidearm flasks Sterile Micropipettes and tips Sterile Macro pipettes and tips Sterilized cigarette tobacco Ethanol Bunsen burner Incubator Saccharomyces cerevisiae (Lab strain yeast) DH5 Alpha E. Coli Matches

9. Procedure I.Yeast 1.Saccharomyces cervevisiae was grown overnight in sterile YEPD media 2.A sample of the overnight culture was added to fresh YEPD media in a sterile sidearm flask 3.The culture was placed in a shaking water bath (30 0 C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of 10 7 cells/ml. 4.The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/ml. 5.Tobacco product fragments were sterilized in an autoclave 6.The selected masses of tobacco products were added to the variable tubes and allowed to sit for 30 minutes 7.After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on 24 plates 8.The plates were incubated at 30 0 C for 48 hours. 9.The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

10. Procedure I. Bacteria 1.E. coli was grown overnight in sterile LB media 2.A sample of the overnight culture was added to fresh LB media in a sterile sidearm flask 3.The culture was placed in a shaking water bath 4. The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/ml. 5.Tobacco product fragments were sterilized in an autoclave 6.The selected masses of tobacco products were added to the variable tubes and allowed to sit for 30 minutes 7.After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on 24 plates 8.The plates were incubated at 30 0 C for 48 hours. 9.The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

11. Yeast Survivorship P-Value: 1.46 x 10-7

12. Yeast Survivorship

13. Bacterial Survivorship P-Value: 1.28 x 10 -7

14. Bacterial Survivorship

15. Statistical Analysis ConcentrationsT-value compared to t-critical 0.01 mg/ml 4.16>3.10 Significant 0.1 mg/ml 7.03>3.10 Significant 1 mg/ml 12.84>3.10 Significant T-critical: 3.10Yeast ConcentrationsT-value compared to t-critical 0.01 mg/ml 3.92>3.10 Significant 0.1 mg/ml 6.45>3.10 Significant 1 mg/ml 9.78>3.10 Significant E. coli

16. Conclusion Since the T-Value of both the Yeast data and the E. coli was greater than 3.10, the alternate hypotheses can be accepted and the null hypotheses can be rejected. It can be concluded that the presence of tobacco fragments significantly altered microbial survivorship

17. Limitations Sterility Tobacco products might not have been given enough time to sit with yeast Yeast may not have been equally spread on the plate

18. Extensions Other pollutants may have been used –Narcotics, smoke, oil Other methods of exposure may have been used –Exposure to burned tobacco Use more replicates and multiple tubes to conform data

19. Sources http://www.adha.org/media/facts/tobacc o.htmhttp://www.adha.org/media/facts/tobacc o.htm http://www.antiproibizionisti.it/public/doc s/thelancet_20070323.pdfhttp://www.antiproibizionisti.it/public/doc s/thelancet_20070323.pdf http://www.aboutmyplanet.com/environ ment/smoking-affects/http://www.aboutmyplanet.com/environ ment/smoking-affects/ https://myxo.css.msu.edu/index.html Special Thanks to Dr. John Wilson