EFFECTS OF SUNSCREEN ON YEAST CELL SURVIVORSHIP

By Peter Koltas, Pittsburgh Central Catholic High School, 9th grader, First year in PJAS.

Problem

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

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.

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

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.

Sunscreens Being Tested

• Coppertone Sport High Performance Sunscreen SPF 30.

• Coppertone Ultraguard Sunscreen SPF 30.• CVS Clear Spray SPF 30.

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.

Purpose

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

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.

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)

Procedure1. Saccharomyces cerevisiae were grown overnight in

sterile YEPD media.

2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask.

3. 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.

4. The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL.

5. Test tubes were made with concentrations of 0%, 0.1%, 1% and 10% sunscreen.(Three replicates, one for each type of sunscreen.)

Test Tube Concentrations

0% 0.10% 1% 10%sterile water 9.9 ml 9.89 ml 9.8 ml 8.9 mlyeast 0.1 ml 0.1 ml 0.1 ml 0.1 mlsunscreen 0 ml 0.01 ml 0.1 ml 1 ml

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.

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

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.

Dunnett’s Tests Results

Test T Value Result0% vs. 0.1% CVS 6.80504 Significant

0% vs. 0.1% Sport 6.113 Significant

0% vs. 0.1% Guard 5.19028 Significant

0% vs. 1% CVS 4.53669 Significant

0% vs. 1% Sport 4.88271 Significant

0% vs. 1% Guard 4.26757 Significant

0% vs. 10% CVS 3.57553 Significant

0% vs. 10% Sport 3.26795 Significant

0% vs. 10% Guard 3.07572 Significant

T Critical is 2.23

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.

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.

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.

Bibliographyhttp://futurescienceleaders.org/researchers2012/2013/03/why-is-yeast-used-inresearch/

http://humantouchofchemistry.com/history-of-sunscreens.htm http://www.randomhistory.com/2009/04/28_sunscreen.html www.coolasuncare.com/sun-science/uva-vs-uvb

http://www.marchelli.it/Uni/OrganismiModello/Yeast/Yeast%20as%20Model%20Genetic%20Organism.pdf

http://www.biology.arizona.edu/cell_bio/tutorials/cell_cycle/cells3.html

http://www.ewg.org/2014sunscreen/the-trouble-with-sunscreen-chemicals/

ANOVAAnova: Single Factor

SUMMARYGroups Count Sum Average Variance

Column 1 4 192 48 0Column 2 4 299 74.75 14.25Column 3 4 341 85.25 67.58333Column 4 4 323 80.75 582.9167Column 5 4 275 68.75 18.25Column 6 4 282 70.5 15Column 7 4 291 72.75 12.25Column 8 4 244 61 18Column 9 4 257 64.25 132.9167Column 10 4 249 62.25 154.25

ANOVASource of VariationSS df MS F P-value F crit

Between Groups4097.525 9 455.2806 4.483682 0.000863 2.210697Within Groups3046.25 30 101.5417

Total 7143.775 39

ANOVA statistical analysisSingle Factor A-Nova

P Value =1.56E-14

Significant

Single Factor A-Nova on isolated 0.1%concentration

P Value= 0.539871

Not Significant

Single Factor A-Nova on isolated 1% concentration

P Value= 0.574469

Not Significant

Single Factor A-Nova on isolated 10% concentration

P Value= 0.614414

Not Significant