1. Nolan Martino Pittsburgh Central Catholic High School Grade 9

2.  The majority of teens males ages 13-18 use aerosol body spray products.  Many teens overuse these products so that their scent will be stronger.  The microbial flora found on the human body has many functions vital to human health.  Does the use of aerosol body spray have any effects on the human skin flora?

3.  4 main ingredients: Ethanol, Difluoroethane, Polyaminopropyl biguanide, and a secret unknown fragrance substance  Popular among male teens

4.  Bacteria that lives on the surface of the skin.  Over a trillion bacteria on our skin.  Usually mutualistic or commensal, but can be harmful if entering the body.  Can stimulate the immune system and promote health.

5.  Gram-positive  Part of the normal skin flora  Grows best in aerobic conditions even though it is anaerobic  Form clusters and can create biofilms

6.  To assess the effects of aerosol body spray products on microbial skin flora

7.  Null Hypothesis: Axe aerosol body spray will have no effect on bacterial survivorship  Alternative Hypothesis: Axe aerosol body spray will significantly reduce bacterial survivorship

8.  Sterile aerosol body spray (Axe Apollo)  Vortex  Metal wire spreader  Ethanol  Bunsen burner  Test tubes  Tube rack  Incubator  Staphylococcus epidermidis  LB agar plates  SDF (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)  LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)  Micropipettes  Sterile pipet tips

9. 1. Bacteria (Staph) was grown overnight in sterile LB 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 (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10⁸ cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL. 5. The experimental variable was mixed with the appropriate amounts of SDF to create concentrations of 0%, 0.1%, 1%, and 10%

10. Tube 1 10% Tube 2 1% Tube 3 0.1% Tube 4 0% Sterile dilution fluid 8.9 mL Microbe0.1 mL Sterile fluid0 mL0.9 mL0.99 mL1.0 mL Axe body spray 1.0 mL0.1 mL0.01 mL0 mL Total Volume 10 mL

11. 6) 100 µL of cell culture was then added to the solutions, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7) The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 9. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB-agar plates. 10. The plates were incubated at 37 C for 24 hours. 11. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

12.  ANOVA Statistical test that compares the means of multiple groups.  Dunnett’s Test A statistical test that compares an experimental group to the control group

13. P-Value=1.3729E-13

14. T-Critical = 2.54 Alpha= 0.05 Aerosol Body Spray ConcentrationT-ValueInterpretation 0.1%1.54 Not significant 1%13.58 Significant 10%16.55 Significant

15. 1. The following volumes of Axe body spray were spread on LB-agar plates to create the desired concentrations: 200 uL (1%), 20uL (0.1%), and 2uL (0.01%) 2. 100 µL aliquots of bacterial suspension from the CONTROL tube (Expt. 1) were spread onto the infused plates. 3. The plates were incubated at 37 C for 24 hours and the resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

16. P-Value= 2.02E-07

17. T-Critical = 2.63 Alpha= 0.05 Aerosol Body Spray AmountT-ValueInterpretation 2 uL0.43 Not significant 20 uL0.83 Not significant 200 uL10.18 Significant

18.  Experiment 1(Liquid Pulse) ◦ Null hypothesis is accepted at the concentration 0.1% but rejected at concentrations 1% and 10% ◦ Beyond a certain level, an increase in concentration of aerosol body spray is generally associated with a decrease in the survivorship of Staph e.  Experiment 2 (Agar Infusion) ◦ Null hypothesis is accepted at the amount 2uL but rejected at amounts 20uL and 200uL ◦ Beyond a certain level, an increase in the amount of aerosol body spray is generally associated with a decrease in the survivorship of Staph e.

19.  At lower concentrations, Axe body spray seems to be safe to use.  Above a concentration of 0.1%, Axe body spray is harmful to healthy skin flora.  The makers of Axe body spray should be careful not to create too highly concentrated products and should give users instructions about how much of the product to use and how often.  People who use Axe body spray should follow recommendations about how to use the product and should consider taking a break from using the spray every once in a while so that their skin flora can recuperate.

20. Limitations  Plating slightly unsynchronized  Only 3 Concentrations of variable tested  Only one exposure time utilized  Only Survivorship assessed Future Studies  Use more types of aerosol body spray products  Use more concentrations  Vary exposure times  Growth curve  Synergistic effects?

21.  http://www.dailymail.co.uk/health/article-2402692/The- hidden-dangers-deodorant-sprays-Headaches-Eczema- Asthma-Even-fatal-heart-problems.html http://www.dailymail.co.uk/health/article-2402692/The- hidden-dangers-deodorant-sprays-Headaches-Eczema- Asthma-Even-fatal-heart-problems.html  http://www.theaxeeffect.com/ http://www.theaxeeffect.com/  http://www.elephantjournal.com/2010/03/your-little-man- on-axe-elizabeth-joseph/ http://www.elephantjournal.com/2010/03/your-little-man- on-axe-elizabeth-joseph/  http://www.natural-skincare-authority.com/axe-deodorant- body-spray.html#top http://www.natural-skincare-authority.com/axe-deodorant- body-spray.html#top  http://www.allure.com/beauty-trends/blogs/daily-beauty- reporter/2013/03/axe-body-spray-life-threatening- allergic-reaction.html http://www.allure.com/beauty-trends/blogs/daily-beauty- reporter/2013/03/axe-body-spray-life-threatening- allergic-reaction.html  http://www.textbookofbacteriology.net/themicrobialworld/N ormalFlora.html http://www.textbookofbacteriology.net/themicrobialworld/N ormalFlora.html

22. Number of Colonies Per Plate Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Mean 0%562755617552621501601 0.1%490505550632563527545 1%503718935164175108 10%0000000

23. Number of Colonies Per Plate Plate 1Plate 2Plate 3Plate 4Plate 5Plate 6Mean 0 mL † 562755617552621501601 2 mL585463642637n/a 582 20 mL552564581560n/a 564 200 mL182118129165n/a 149 † Data from Experiment 1 n/a = not applicable

25. ANOVA: Single Factor SUMMARY GroupsCountSumAverageVariance 0 mL63608601.33337658.667 2 mL42327581.756931.583 20 mL42257564.25149.5833 200 mL4594148.5901.6667 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups596519.9319884044.72492.02E-073.343889 Within Groups62241.83144445.845 Total658761.817