Cigarette Leachate Effects on Microbial Survivorship

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 • Yeast • Saccharomyces cervevisiae was grown overnight in sterile YEPD media • A sample of the overnight culture was added to fresh YEPD media in a sterile sidearm flask • The culture was placed in a shaking water bath (300 C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of 107 cells/ml. • The cell culture was diluted in sterile dilution fluid to a concentration of approximately 103 cells/ml. • Tobacco product fragments were sterilized in an autoclave • The selected masses of tobacco products were added to the variable tubes and allowed to sit for 30 minutes • After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on 24 plates • The plates were incubated at 300 C for 48 hours. • The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

10. Procedure I. Bacteria • E. coli was grown overnight in sterile LB media • A sample of the overnight culture was added to fresh LB media in a sterile sidearm flask • The culture was placed in a shaking water bath • The cell culture was diluted in sterile dilution fluid to a concentration of approximately 103 cells/ml. • Tobacco product fragments were sterilized in an autoclave • The selected masses of tobacco products were added to the variable tubes and allowed to sit for 30 minutes • After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on 24 plates • The plates were incubated at 300 C for 48 hours. • 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 Yeast T-critical: 3.10 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/tobacco.htm • http://www.antiproibizionisti.it/public/docs/thelancet_20070323.pdf • http://www.aboutmyplanet.com/environment/smoking-affects/ • https://myxo.css.msu.edu/index.html • Special Thanks to Dr. John Wilson