Vitamin E Attenuation on Heat and UV Stressed Yeast Cells

1. Vitamin E Attenuation on Heat and UV Stressed Yeast Cells Joseph H. Gault February 5, 2011 Central Catholic High School

2. Ultra Violet Light • Electromagnetic radiation • The wavelength is shorter than that of visible light • The shorter wavelength has greater energy • The sun produces UV light • Responsible for sunburn and some cancers • Alters and damages DNA

3. Heat Stress • Can cause many problems in people, including heat exhaustion, heat stroke, heat cramps, heat rash, etc • These conditions can be cured in a number of ways: • Hydration • Loose Clothing • Skin ointment • Vitamin E

4. Vitamin E • Antioxidant • Protects skin from UV damage • Minimizes cell damage from free radicals • May protect the heart in lower doses • Found in leafy green vegetables, olives, and various nuts and seeds

5. Saccharomyces cerevisiae(Yeast) • Eukaryotic microorganism • Unicellular • 3–4 µm diameter • Used in baking and production of alcoholic beverages • The most studied cellular model in research • Cell cycle is similar to human cells • Comparable DNA replication, recombination, cell division and metabolism

6. Purpose • The purpose of this experiment was to determine if the antioxidant Vitamin E can increase the survivorship of heat and UV stressed yeast cells.

7. Null Hypothesis • The vitamin E concentrations will not significantly affect UV-stressed and heat stressed yeast survivorship.

8. Materials • Test tubes • Test tube rack • Micropipettes • Pipette tips • Yeast • (Saccharomyces • cerevisiae) • YEPD Agar Plates • YEPD Media (0.5% yeast, • 2% Glucose, 2% Peptone) • Vitamin E • UV Lamp/hood • Proper safety equipment • Water • Spreader bar • Vortex • SDF • UV Safety Glasses • Thermometer • Water • Hot Plates • Beakers

9. Procedure • Saccharomyces cerevisiae was grown overnight in sterilized YEPD media. • A sample of the overnight cultures was added to fresh media in a sterile sidearm flask. • The culture of yeast was incubated at 30°C until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells per mL. • The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells per mL.

10. UV Stress Procedure • The following tubes were created: • 0.1 mL of each suspension was plated onto YEPD agar plates (54 total plates). • The plates were exposed to UV light for the following time intervals: • 0 Seconds • 30 Seconds • 60 Seconds

11. Heat Stress Procedure • Equivalent tubes were submerged in water baths for fifteen minutes at the following temperatures: • 32°C • 42°C • 52°C • 0.1mL of each suspension was plated onto YEPD agar plates (54 total plates) • Plates were incubated at room temperature for 48 hours • Colonies were counted. Each colony was assumed to have arisen from one cell.

12. 100 uL 100 uL 100 uL 107 cells/mL (yeast) Dilution Procedure 105 cells/mL 103 cells/mL 102 cells

13. Data - UV

14. Data - UV

15. Data - UV

16. Data - Heat

17. Data - Heat

18. Data - Heat

19. Vitamin E Attenuation of UV Stress P-Value: 4.78*10-09 P-Value: 5.67*10-17 P-Value: .097

20. Vitamin E Attenuation of Heat Stress P-Value: 1.40*10-12 P-Value: 3.07*10-12 P-Value: 0.884

21. Results • There was no significant variance in survival of the control groups treated with vitamin E. • There was a significant variation in survivorship of cells exposed to UV and not treated with vitamin E. • There was significant variation in survivorship of cells exposed to heat and not treated with vitamin E. • There was significant variation in survivorship of cells when exposed to the various temperatures and treated with vitamin E.

22. Conclusions • The ANOVA indicated significant variation in the survivorship of the yeast colonies. • Therefore, the null hypothesis was rejected. • Vitamin E significantly affects yeast survivorship when exposed to heat and UV stress.

23. Limitations/Extensions • Lag time • Use different concentrations of Vitamin E • Use different types of antioxidants (Lycopene, Vitamin A, Vitamin C) • Expose to varying amounts of UV light • Expose to a wider range of temperatures • Increase sample size

24. Sources • "Differential Response to UV Stress and DNA Damage during the Yeast Replicative Life Span - Kale - 1998 - Developmental Genetics." Wiley Online Library. Web. 25 Jan. 2011. <http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1520-6408(1996)18:23.0.CO;2-8/abstract>. • "Heat Stress." Welcome To Web.princeton.edu. Web. 29 Jan. 2011. <http://web.princeton.edu/sites/ehs/heatstress/heatstress.htm>. • "Safety and Health Topics: Heat Stress." Occupational Safety and Health Administration - Home. Web. 25 Jan. 2011. <http://www.osha.gov/SLTC/heatstress/>. • "Vitamin E: MedlinePlus Medical Encyclopedia." National Library of Medicine - National Institutes of Health. Web. 25 Jan. 2011. <http://www.nlm.nih.gov/medlineplus/ency/article/002406.htm>. • "Vitamin E." Office of Dietary Supplements (ODS). Web. 25 Jan. 2011. <http://ods.od.nih.gov/factsheets/vitamine/>. • "Vitamin E." Wikipedia, the Free Encyclopedia. Web. 25 Jan. 2011. <http://en.wikipedia.org/wiki/Vitamin_E>. • Web. 29 Jan. 2011. <http://www.ncbi.nlm.nih.gov/pubmed/12443690>. • "WHFoods: Vitamin E." The World's Healthiest Foods. Web. 29 Jan. 2011. <http://www.whfoods.com/genpage.php?tname=nutrient&dbid=111>.