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

Problem Cologne is used worldwide with unknown effects on 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 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

Cologne being tested Tarragon Noir Jacose Blue Hot Thrills Jordache Men No. 37

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.

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

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.

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

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)

Test Tube Concentrations 0% 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

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.

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%

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

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

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

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 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

Bibliography http://davidmlane.com/hyperstat/B112114.html http://wiki.yeastgenome.org/index.php/What_ar e_yeast%3F http://www.ewg.org/research/not-so-sexy https://statistics.laerd.com/statistical- guides/one-way-anova-statistical-guide.php http://textbookofbacteriology.net/normalflora.ht ml https://www.wavemetrics.com/products/igorpro /dataanalysis/statistics/tests/statistics_pxp11.ht m

Single Factor A-Nova