1. Cologne Effects on Yeast Survivorship
Joshua Egan
10th grade, Pittsburgh Central Catholic High School, 2nd year in PJAS

2. Problem
Cologne is used worldwide with unknown effects on 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
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. Cologne being tested Tarragon Noir Jacose Blue Hot Thrills
Jordache Men No. 37

6. Cologne Ingredients Common ingredients between all four brands:
Acetone, Benzaldehyde, Benzyl Acetate, Benzyl Alcohol, Ethyl Acetate, Limonene, Linalool, Methylene Chloride, a-Pinene, g-Terpinene, a- Terpineol, etc.

7. Purpose
The purpose of this experiment is to test the effects of four commercial brands of cologne on microbial survivorship, specifically Saccharomyces cerevisiae.

8. Hypothesis
Null hypothesis: None of the brands of cologne will have a significant effect on yeast cell survivorship
Alternate hypothesis: The brands of cologne will have a significant negative effect on yeast cell survivorship.

9. Materials Saccharomyces cerevisiae Jordache Men No. 37 brand cologne
Micropipettes
Tarragon Noir brand cologne
Micro Rack
Sterile Water
Micro Tubes
Sterile Pipette Tips
Agar Plates
48 YEPD Agar plates (1% yeast extract, 2% glucose, 1.5% agar)
Vortex
Incubator
YEPD Media (1% yeast extract, 2% peptone, 2% glucose)
Spreader Bars
Ethanol
Sterile dilution fluid (10 mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)
Bunsen Burner
Hot Thrills brand cologne
Jacose Blue brand cologne

10. 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 107 cells/mL
The cell culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL
Test tubes were made with concentrations of 0%, 0.1%, 1%, and 10% cologne. (3 replicates for each of four concentrations for each of four brands – 48 plates)

11. Test Tube Concentrations0%
0.1% 1%
10%
Sterile water
9.9 mL
9.89 mL
9.8 mL
8.9 mL
Yeast
0.1 mL
Cologne
0 mL
0.01 mL
1 mL

12. Procedure cont.
6. The tubes were incubated at room temperature for 15 minutes 7. Tubes were vortexed and 0.1 mL 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.

13. 120
110
P-value for all data: 2.73 X 10-7 (significant)
105
106 97
100 85 83 80 80 76 70 69 60 60 53 38 40 20 0%
0.1% 1%
10%

14. A-Nova/Dunnett's Test A-Nova: Dunnett's Test:
Statistical test that allows for the comparison of means of different groups, to determine significant variation
Utilize p-values as measures of significance
Confidence interval of the likelihood of seeing the same effect produced by the same variable with replication
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

15. Dunnett’s Test Results
T Value
Result
0% vs. 0.1% Tarragon Noir
1.265
Not significant
0% vs. 1% Tarragon Noir
1.518
0% vs. 10% Tarragon Noir
3.643
Significant
0% vs. 0.1% Jacose Blue
0.278
0% vs. 1% Jacose Blue
1.888
0% vs. 10% Jacose Blue
2.777
0% vs. 0.1% Jordache Men
0.290
0% vs. 1% Jordache Men
1.955
0% vs. 10% Jordache Men
2.969
0% vs. 0.1% Hot Thrills
0.771
0% vs. 1% Hot Thrills
2.372
0% vs. 10% Hot Thrills
3.380
t-crit = 2.75

16. Interpretation of Results
The null hypothesis can be rejected
The alternate hypothesis can be accepted
All brands of cologne produced a significant negative effect on the yeast survivorship
After running the Dunnett’s Test, it was found that the significant effect for all brands against the control was at 10% concentration

17. 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

18. Extensions
Addition of more microbial models such as Staphylococcus and E-coli
Other brands of cologne 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

19. Bibliography http://davidmlane.com/hyperstat/B112114.html
e_yeast%3F
guides/one-way-anova-statistical-guide.php ml
/dataanalysis/statistics/tests/statistics_pxp11.ht m

20. Single Factor A-Nova