Effects of Axe Body Spray on Staph and Yeast Survivorship Michael Halahurich 11th grade, Pittsburgh Central Catholic High School, 2nd year in PJAS

Problem Axe Body Spray is a widely used product popular especially among adolescents with unknown effects on microbial life

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 medicine, food, and skin products may have unintended effects on the body’s flora populations and their functions

Yeast Saccharomyces cerevisiae Easy to manipulate Most commonly studied cell Resembles advanced eukaryotic cells such as human cells in metabolism, reproduction, etc. Used in this study to represent eukaryotic skin flora

Staph Staphylococcus epidermidis Widely studied Gram-positive bacterium Part of the normal skin microflora Various positive effects for the body Inhibition and killing of nonindigenous species

Axe Body Spray Being Tested Axe Twist Body Spray Components Alcohol Denat., Hydrofluorocarbon 152A, Fragrance (Parfum), Polyaminopropyl Biguanide Stearate.

Purpose The purpose of this experiment is to determine the effects of Axe Body Spray on two different forms of microbial life—Staphylococcus epidermidis and Saccharomyces cerevisiae.

Hypothesis Null hypothesis: Axe Body Spray will not have a significant effect on staph and yeast survivorship. Alternate hypothesis: Axe Body Spray will have a significant effect on staph and yeast survivorship.

Materials Saccharomyces cerevisiae Axe Twist Body Spray Staphylococcus epidermidis Sterile Water Micropipettes Sterile Pipette Tips Micro Rack 60 YEPD Agar plates (1% yeast extract, 2% peptone 2% glucose, 1.5% agar) Micro Tubes Agar Plates YEPD Media (1% yeast extract, 2% peptone, 2% glucose) Vortex Incubator Spreader Bars Sterile dilution fluid (10 mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) Ethanol Bunsen Burner

Procedure Microbes 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 incubated at 30 degrees Celsius 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% Axe. (5 replicates for each of the four concentrations – 20 plates per microbe)

Test Tube Concentrations 0% 0.1% 1% 10% Sterile water 9.9 mL 9.89 mL 9.8 mL 8.9 mL Microbe 0.1 mL Axe 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.

Procedure (Agar Infusion) Steps 1-4 from the previous procedure were repeated Ten agar plates were treated with a “low concentration” of Axe (20 microliter of Axe, 180 microliters of water) Ten agar plates were treated with a “high concentration” of Axe (200 microliters of Axe) Plates were incubated for 15 minutes Five “high concentrations” and five “low concentrations” were treated with 100 microliters from the yeast control tube Five “high concentrations” and five “low concentrations” were treated with 100 microliters from the staph control tube 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

Agar Infusion Concentration Low High Sterile water 0.18 mL 0 mL Axe 0.02 mL 0.2 mL Note: These amounts were directly pipetted onto the respective agar plates

A-Nova/Dunnett's Test A-Nova: Statistical test that allows for the comparison of means of different groups to determine significant variation Dunnett's Test: A follow up test used to find out which variable groups produced significant variation compared to a control

P-value: 3.94E-13 P-value: 4.8E-14

Dunnett’s Test Results T Value Result (YEAST) 0% vs. 0.1% Axe 5.13 Significant (YEAST) 0% vs. 1% Axe 17.27 (YEAST) 0% vs. 10% Axe 22.61 (STAPH) 0% vs. 0.1% Axe 4.40 (STAPH) 0% vs. 1% Axe 19.54 (STAPH) 0% vs. 10% Axe 24.12 t-crit = 3.239

P-value: 8.11E-06 P-value: 1.74E-08

Dunnett’s Test Results (Agar Infusion) T Value Result (YEAST) Low Concentration Axe 2.91 Not significant (YEAST) High Concentration Axe 8.39 Significant (STAPH) Low Concentration Axe 0.55 (STAPH) High Concentration Axe 13.22 t-crit = 3.885

Interpretation of Results The null hypothesis can be rejected The alternate hypothesis can be accepted Axe Body Spray produced a significant negative effect on yeast and staph survivorship Statistical analyses supported a significant effect at all concentrations

Interpretation of Results (Agar Infusion) The null hypothesis can be rejected for the “high” concentrations of Axe The alternate hypothesis can be accepted for the “high” concentrations of Axe Exposure to Axe Body Spray via agar infusion produced a significant negative effect on yeast and staph survivorship Statistical analyses supported significant effects at high concentrations

Limitations Slight positioning differences in the incubation process Slightly desynchronized plating Limited concentration exposures Only survivorship tested, not growth or other parameters Study does not account for other factors that might affect the skin microbial flora

Extensions Addition of more microbial models such as E-coli Would allow for a gram-positive vs. gram- negative comparison Other brands of body spray could be used for further analysis Different concentrations of the variable could be tested

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 http://www.bioquell.com/en-us/resources-and- support/microbiology/staphylococcus-epidermis/

Single Factor A-Nova (Yeast) Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 1410 282 1420 Column 2 1090 218 107.5 Column 3 374 74.8 26.7 Column 4 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 250221.4 3 83407.13 214.6625 3.94E-13 3.238872 Within Groups 6216.8 16 388.55 Total 256438.2 19  

Single Factor A-Nova (Staph) Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 1628 325.6 770.8 Column 2 1339 267.8 779.2 Column 3 343 68.6 179.3 Column 4 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 364385.8 3 121461.9 280.9505 4.8E-14 3.238872 Within Groups 6917.2 16 432.325 Total 371303 19  

Single Factor A-Nova (Yeast Agar Infusion) Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 1410 282 1420 Column 2 1108 221.6 1622.3 Column 3 538 107.6 196.3 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 78432.53 2 39216.27 36.32705 8.11E-06 3.885294 Within Groups 12954.4 12 1079.533 Total 91386.93 14  

Single Factor A-Nova (Staph Agar Infusion) Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 1628 325.6 770.8 Column 2 1580 316 1136 Column 3 478 95.6 363.3 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 169280.5 2 84640.27 111.8545 1.74E-08 3.885294 Within Groups 9080.4 12 756.7 Total 178360.9 14