1. T HE E FFECTS OF O LIVE O IL A GAINST C IGARETTE S MOKE ON Y EAST Peter Wagstaff

2. P ROBLEM Cigarette smoking is the leading cause of preventable death in the United States. Quitting is ideal but not always easy or expedient.

3. C IGARETTE S MOKE Cigarette smoke contains thousands of chemicals, many of them toxic or cancer causing, such as arsenic and tar An estimated 480,000 deaths are caused by cigarettes every year in the United States. Cigarettes are the leading cause of preventable death in the United States

4. O LIVE O IL Contains Oleic Acid Unsaturated fat Unsaturated fats are cited as a lung supporting nutrient by the National Lung Association Previous studies have reported that diets high in unsaturated fats can shield against some effects of smoking However, other studies have indicated that the burning of cooking oils can increase risk for lung cancer

5. W HY Y EAST (S ACCHAROMYCES CERERISAE ) Yeast is used as a model because: Eukaryotic, just like human cells Most studied cell Many characteristics like nutrition and cell growth are similar to human cells Aerobic

6. P URPOSE Can unsaturated fats (olive oil) reduce the negative effects of cigarette smoke on yeast? Will oil alone affect yeast survivorship? Will smoke alone affect yeast survivorship?

7. H YPOTHESIS Null: The olive oil and cigarette smoke will NOT have a significant effect on yeast survivorship, individually or synergistically. Alternative 1: The presence of olive oil or cigarette smoke will have a significant individual effect on yeast survivorship. Alternative 2: Olive oil will significantly increase the survivorship of smoke stressed yeast.

8. M ATERIALS Saccharomyces cerevisiae (Yeast) YEPD agar plates (1% yeast extract, 1.5% agar, 2% glucose) Aldi brand virgin olive oil Sterile micropipettes and tips (200ml) Sharpie Sterile dilution fluid (100mM KH 2 PO 4. 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl) Sterile spreader bars Ethyl alcohol Burner Marlboro cigarettes Stop watch Smoke Chamber Incubator Spectrophotometer

9. P ROCEDURE 1. A culture Saccharomyces cerevisiae was grown. 2. The culture was incubated at 30 degrees Celsius until a density of 50 Klett spectrophotometer units were reached. This represents a density of approximately 10 ⁷ cells/mL. 3. The culture was diluted with sterile dilution fluid to a concentration of 10^3 cells/mL

10. P ROCEDURE 4. 100μl of the cell suspension was pipetted into olive oil infused YEPD agar plates Agar infusion : After autoclaving YEPD agar, sterile olive oil was mixed with the molten agar to create the following infused plates; 0%, 0.1%, 1%, and 10% 5. Six plates of each concentration were taped to the top of the smoke chamber and exposed to 0, 45, 90, and 130 seconds of smoke

11. P ROCEDURE 7. A fresh cigarette was lit and fitted into the socket for each exposure, and the air pump was pumped every three seconds for consistent airflow. 8. The plates were covered and incubated at 30 degrees Celsius for 72 hours and the resulting colonies were quantified.

12. D OCUMENTATION OF S MOKE C HAMBER

15. ANOVA Single Factor Stat test comparing the means of multiple groups Alpha of 0.05 is selected Two Factor Can reveal synergistic affects of two variables

16. S MOKE E FFECTS ON Y EAST S URVIVORSHIP Number of colonies Overall P-value: 0.00171 Average Yeast Survivorship Smoke Exposure [seconds]

17. O IL E FFECT ON Y EAST S URVIVORSHIP Number of colonies Average Yeast Survivorship Overall P-value: 4.24E-06 Concentration[Oil]

18. D UNNETT ’ S T EST The Dunnett's test is a follow up to the ANOVA that quantifies the difference between a variable and control group to find significant variation.

19. D UNNETT ' S T EST GroupT ValueSignificance 0.1% Oil2.432Not Significant 1% Oil0.024Not Significant 10% Oil-4.936Significant Oil did not begin to have a significant effect on yeast until the concentration reached 10% T-Crit = 3.098391

20. S MOKE AND O IL E FFECTS ON S TRESSED Y EAST Number of colonies Average Yeast Survivorship Overall P-value: 0.017711

21. Did olive oil have an effect by itself? P-Value 4.24E-6 Significant Did cigarette smoke reduce yeast survivorship? P-Value 0.00171 Significant Did oil increase survivorship in smoke stressed yeast? P-Value 0.017711 Significant

22. C ONCLUSION The null hypothesis was rejected. Both variables had individual effects as well as a synergistic effect. Both oil and smoke harmed the yeast, however oil created a shielding affect at high concentrations on smoke stressed yeast.

23. L IMITATIONS /E XTENSIONS Limitations Lag time between smoke exposures could have created unwanted variance Yeast should have been exposed more immediately after plating Extensions More replicates More exposure times and concentrations of oil A more sophisticated smoke chamber

24. R EFERENCES [http://atvb.ahajournals.org/content/21/6/1029.lon g] [http://www.ncbi.nlm.nih.gov/pubmed/10618003] [http://cancergrace.org/lung/2007/11/27/cooking- oil-fumes-and-lcins/]

25. S INGLE F ACTOR ANOVA FOR S MOKE E FFECTS GroupsCountSumAverageVariance Column 1648180.1666747.76667 Column 2645575.8333352.56667 Column 3642570.8333342.56667 Column 4638163.525.5 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups921.83333307.27787.298760.001713.098391 Within Groups8422042.1 Total1763.83323

26. S INGLE F ACTOR ANOVA FOR O LIVE O IL E FFECTS GroupsCountSumAverageVariance Column 1648180.1666747.76667 Column 2658196.83333282.5667 Column 3648280.33333223.4667 Column 4627846.333339.866667 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups8101.532700.519.163814.24E-063.098391 Within Groups2818.33320140.9167 Total10919.8323

27. 2 F ACTOR ANOVA S UMMARY Source of VariationSSdfMSFP-valueF crit Sample14810.8634936.95563.927773.46E-212.718785 Columns1598.6153532.87156.900060.0003432.718785 Interaction1677.5949186.39932.4136520.0177111.999115 Within6178.1678077.22708