1. By : Phillip DeRenzo 9th Grade Central Catholic High School

2. Introduction People all over the world smoke. Most of them know that smoking is harmful to their bodies, but do they know the damaging effects that smoke has on their cells? In addition, what effects does secondhand smoke have on flora in the body? (Ecology) Two experiments were performed to explore smoke toxicity. Cellular Model – Yeast Smoke Products- Cigarettes (Garcia y Vega: English Coronas)/Cigars (Marlboro: Filtered Cigarettes)

3. Cigarette/Cigar Smoke Cigarette/Cigar smoke has harmful effects- releases toxins, irritants, etc. Main pollutants are acetone, carbon monoxide, arsenic, benzene, butane, cyanide, nicotine, lead, formaldehyde, and many others

4. Background of Cigar vs. Cigarette Smoke Past studies focus on adverse effects on human body. Cigars have shown to be more damaging than cigarettes. Higher levels of carcinogens These carcinogens can induce abnormal cell cycling (cancer). Cigarettes appear to be more addictive. Cigarette smokers inhale more deeply, creating more respiratory tree problems (lungs). Upon inhalation, nicotine can reach the brain 10 seconds.

5. Secondhand Smoke Smoke that is inhaled involuntarily, or passively, by someone who is not smoking. A child of a smoker is more likely to have asthma and other breathing problems because of secondhand smoke inhalation.

6. Yeast Eukaryotic, unicellular fungi. Has been widely used in genetics and cell biology, largely because the cell cycle in a yeast cell is very similar to the cell cycle in higher organisms (human).

7. Experimental Cell Model Saccharomyces cerevisiae- a common yeast Great ease of manipulation in the laboratory (single-celled microbe) Similar biochemistry, genetics, cell cycle to other eukaryotes, including human cells MOST STUDIED CELL IN THE WORLD Colonies Petri Dish Agar

8. Purpose Do cigarettes or cigars affect the survival of microbial flora? Can secondhand smoke persist on a surface and harm microbial flora? Does increased exposure increase the damaging effects of smoke on microbial flora?

9. Hypotheses Null Hypothesis : Smoking product exposure will not reduce survivorship of yeast. Alternate Hypothesis : The smoking products will reduce survivorship of yeast.

10. Materials YEPD agar plates (1% yeast extract, 2% glucose, 1.5% agar) YEPD media (1% yeast extract, 2% peptone, 2% glucose) Sterile capped test tube with sterile dilution fluid (SDF) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) Klett Spectrophotometer Incubator Saccharomyces cerevisiae- a common yeast Micropipettes Permanent Marker Spreader Bar Ethanol Vortex Lighter Box Cigars (Garcia y Vega: English Coronas) Cigarettes (Marlboro: Filtered Cigarettes)

11. Procedure (1 st Experiment) 1. Saccharomyces cerevisiae 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 an incubator (30 o C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of 10 7 cells/mL. 4. The cell suspension was serially diluted to a density of approximately 10 3 cells/mL 5. The tubes were vortexed and then 0.1 mL aliquots were spread onto YEPD plates. 6. Sets of 8 plates were taped to the lid of a box, facing down toward the bottom of the box, and the lids removed. 7. A cigarette or cigar was lit and set it at the bottom of the box. For a time exposure of 1 and 3 minutes, the plates were exposed to the smoke rising from the cigarette lying at the bottom of the box. 8. After exposure, the plates were removed from the box and their lids were put back on. 9. The plates were incubated for 48 hours at 32C. 10. The resulting colonies were counted. Each colony assumed to have arisen from one cell.

12. Procedure (Infused Smoke) 1. 16 YEPD agar plates were selected. 2. 8 of them at a time were taped to the lid of a box, facing down toward the bottom of the box. 3. The lids of the plates were removed, allowing the plates to be exposed to smoke. 4. A cigarette or cigar was lit and set on the bottom of the box. 5. For 10 minutes, the agar plates were exposed to the smoke rising from the cigarette lying at the bottom of the box. 6. When the 10 minutes were over, the plates were removed and covered. 7. 0.1mL aliquots from the 10 3 yeast cell suspension were spread onto the plates 8. The 16 plates were placed in a 32C incubator for 48 hours. 9. The resulting colonies were counted. Each colony assumed to have arisen from one cell.

13. Cigarette Smoke Effects Exposure Time Number Of Colonies

14. Cigar Smoke Effects Exposure Time Number Of Colonies p-value = 0.000406

15. Smoke Infused Agar Effects Exposure Time Number Of Colonies p-value = 2.68E-06

16. Anova (Cigarette) Anova: Single Factor for Cigarette SUMMARY GroupsCountSumAverageVariance Column 18895111.875228.125 Column 287209042.85714 Column 3840050114.2857 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups15752.0827876.04261.329081.7E-093.4668 Within Groups2696.87521128.4226 Total18448.9623

17. Anova (Cigar) Anova: Single Factor for Cigar SUMMARY GroupsCountSumAverageVariance Column 18895111.875228.125 Column 2857071.25469.6429 Column 3849061.25791.0714 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups11502.0825751.04211.588310.0004063.4668 Within Groups10421.8821496.2798 Total21923.9623

18. Anova (Infused Agar) Anova: Single Factor for Infused SUMMARY GroupsCountSumAverageVariance Column 18895111.875228.125 Column 2857571.875349.5536 Column 3840550.625345.9821 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups1547527737.525.130982.68E-063.4668 Within Groups6465.62521307.8869 Total21940.6323

19. Dunnetts’ Test (Cigarette) VariableT ValueInterpretation 60 Seconds3.2Insignificant (Accept Null) 180 Seconds9.1Significant (Reject Null) T Crit = 4.0

20. Dunnetts’ Test (Cigar) VariableT ValueInterpretation 60 Seconds3.8Insignificant (Accept Null) 180 Seconds4.7Significant (Reject Null) T Crit = 4.0

21. Dunnetts’ Test (Infused Agar) VariableT ValueInterpretation Cigarette Smoke Infused Agar Plates 4.5Insignificant (Reject Null) Cigar Smoke Infused Agar Plates 7.0Significant (Reject Null) T Crit = 3.77

22. Conclusion The null hypothesis that the exposure and time of exposure to cigar or cigarette smoke will not effect the growth of yeast was accepted for 60 seconds of exposure. The null hypothesis was rejected for the exposure times of 180 seconds cigarette and cigar. The null hypothesis was rejected for the cigar and cigarette smoke infused agar experiment.

23. Limitations and Extensions Smoke might not have gone directly to the cells Trials could have gone over/under the time of exposure Have a team of people synchronize the spread plating More controlled smoke chamber for exposure More variations of exposure times

24. Sources Scientific American Magazine http://ezinearticles.com/?Which-Is-Worse-For-You,- Cigarettes-Or-Cigars?&id=1181914 http://ezinearticles.com/?Which-Is-Worse-For-You,- Cigarettes-Or-Cigars?&id=1181914 http://quitsmoking.about.com/od/tobaccostatistics/a /CigaretteSmoke.htm http://quitsmoking.about.com/od/tobaccostatistics/a /CigaretteSmoke.htm http://www.quitsmokingsupport.com/cigars.htm