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)
Procedure
1.
2.
3.
4.
5.
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.)
Test Tube Concentrations
0%
sterile water
yeast
sunscreen
9.9 ml
0.1 ml
0 ml
0.10%
1%
10%
9.89 ml 9.8 ml
8.9 ml
0.1 ml
0.1 ml
0.1 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
0% vs. 0.1% CVS
0% vs. 0.1% Sport
0% vs. 0.1% Guard
0% vs. 1% CVS
0% vs. 1% Sport
0% vs. 1% Guard
0% vs. 10% CVS
0% vs. 10% Sport
0% vs. 10% Guard
T Value Result
6.80504 Significant
6.113 Significant
5.19028 Significant
4.53669 Significant
4.88271 Significant
4.26757 Significant
3.57553 Significant
3.26795 Significant
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.
Bibliography
http://futurescienceleaders.org/researchers2012/2013/03/w
hy-is-yeast-used-inresearch/
http://humantouchofchemistry.com/history-ofsunscreens.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/c
ells3.html
http://www.ewg.org/2014sunscreen/the-trouble-withsunscreen-chemicals/
ANOVA
Anova: Single Factor
SUMMARY
Groups
Count
Column 1
4
Column 2
4
Column 3
4
Column 4
4
Column 5
4
Column 6
4
Column 7
4
Column 8
4
Column 9
4
Column 10
4
ANOVA
urce of VariationSS
Between Groups
4097.525
Within Groups
3046.25
Total
7143.775
Sum
Average Variance
192
48
0
299
74.75
14.25
341
85.25 67.58333
323
80.75 582.9167
275
68.75
18.25
282
70.5
15
291
72.75
12.25
244
61
18
257
64.25 132.9167
249
62.25
154.25
df
9
30
39
MS
455.2806
101.5417
F
4.483682
P-value
0.000863
F crit
2.210697
ANOVA statistical analysis
Single Factor ANova
P Value =
1.56E-14
Significant
Single Factor AP Value=
Nova on isolated
0.539871
0.1%concentration
Not Significant
Single Factor ANova on isolated
1% concentration
P Value=
0.574469
Not Significant
Single Factor AP Value=
Nova on isolated
0.614414
10% concentration
Not Significant