1. Effects of Sunscreen on Yeast cell Survivorship
By Peter Koltas, Pittsburgh Central Catholic High School, 9th grader, First year in PJAS.

2. Problem
Many people all over the world use sunscreen often and nobody knows its affects on the skin microflora.

3. Microbial Flora
Little is known about the association between humans and their flora
Effects are mutualistic, parasitic, pathogenic, and commensal
Perform functions beneficial to the host, including the manufacture of essential vitamins, and the prevention of colonization by undesirable microbes.
Human foods, supplements, medicinal, and skin products, may have unintended effects on the flora populations and their functions.

4. Yeast Saccharomyces cerevisiae. Easy to manipulate in laboratories.
Most commonly studied cell.
Has similar reproduction, metabolism, and chemistry as other more advanced eukaryotic cells, like human cells.
Used in this study to represent eukaryotic skin flora

5. Sunscreen Two types of ultraviolet light.
Long wave: UVA
short wave: UVB
UVA rays can age our skin and UVB rays can burn our skin.
UVB blockers: Cinnamates, salicyclates, and anthranilates.
UVA blockers: benzophenones, organic compounds, oxybenzone, dibenzoyl methane.

6. Sunscreens Being Tested
Coppertone Sport High Performance Sunscreen SPF 30.
Coppertone Ultraguard Sunscreen SPF 30.
CVS Clear Spray SPF 30.

7. Sunscreen Ingredients
All three sunscreens contain homoslate(10%), octisalates(5%), oxybenzones(4%), avobenzones(3%), and octocrylene (2%) at the same concentrations.
All of the sunscreens contain denatured alcohol, acrylates, stearoxytrimethsilane, glycerin, diethylhexyl syrungylidenemalonate, tocopherol (Vitamin E), fragrance retinyl palmitate (Vitamin A Palmitate), and caprylic triglyceride are varying concentrations.

8. Purpose
The purpose of this experiment is to test the effects of three commercial sunscreens, SPF 30, on microbial survivorship, specifically Saccharomyces cerevisiae.

9. Hypothesis
Null hypothesis: None of the sunscreens SPF 30 will have an effect on yeast cell survivorship.
Alternate hypothesis: All sunscreens SPF 30 will have significant effects on yeast survivorship in all tested sunscreens in all tested concentrations.

10. Materials Saccharomyces cerevisiae Micropipettes Micro Rack
Micro Tubes
Agar Plates
Vortex
Incubator
Spreader Bars
Ethanol
Bunsen Burner
CVS brand sunscreen SPF 30
Sport brand sunscreen SPF 30
Guard brand sunscreen SPF 30
Sterile water
Sterile pipette tips
48 YEPD Agar plates (1% yeast extract, 2% glucose, 1.5% agar)
YEPD Media (1% yeast extract, 2% peptone, 2% glucose)
sterile dilution fluid (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, 0.1 mM CaCl2, 100mM NaCl)

11. Procedure
Saccharomyces cerevisiae were grown overnight in sterile YEPD media.
A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
The culture was placed in an incubator (30C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^7 cells/mL.
The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL.
Test tubes were made with concentrations of 0%, 0.1%, 1% and 10% sunscreen.(Three replicates, one for each type of sunscreen.)

12. Test Tube Concentrations

13. Procedure cont.
6. The tubes were incubated at room temperature for 15 minutes. 7. Tubes were vortexed and 0.1mL aliquots were plated onto YEPD agar. 8. Plates were incubated at 30 degrees Celsius for two days and colonies were counted. Each colony was assumed to have arisen from a single cell.

15. ANOVA Single factor ANOVA
Statistical test that allows for the comparison of means of different groups, to determine significant variation
Single factor ANOVA
Utilizes p-values as measure of significance
p>0.05: not significant
p<0.05: significant

16. Dunnett’s Test
A test used to find out which variable groups produced significant variation compared to a control.
If T-value is greater than the T critical, variations are considered significant.

17. Dunnett’s Tests Results
T Critical is
2.23

18. Interpretations of Results.
Null hypothesis can be rejected.
Alternate hypothesis can be accepted.
All sunscreens at all concentrations produced significant positive effects on yeast survivorship (growth).
All sunscreens had similar effects at given concentrations.

19. Limitations and Inconsistencies
Slight positioning differences in the incubation process
Slightly desynchronized plating
Only one time of exposure (liquid pulse)
Limited concentration exposures
Only survivorship tested, not growth or other health parameters
Study does not account for other factors that might affect the skin microbial flora.

20. Extensions
Addition of more microbial models such as Staphylococcus and E-coli.
Other brands of sunscreen and different SPF’s could be used for further analysis.
Different concentrations of the variable could be tested.
Varied exposure times
Agar plate infusion
Growth curve analysis
Further statistical analysis could be done on the current data.

21. Bibliography

22. ANOVA

23. ANOVA statistical analysis
Single Factor A-Nova
P Value =
1.56E-14
Significant
Single Factor A-Nova on isolated 0.1%concentration
P Value=
Not Significant
Single Factor A-Nova on isolated 1% concentration
P Value=
Single Factor A-Nova on isolated 10% concentration
P Value=