Effects of Aerosol Body Spray Products

on Skin FloraNolan Martino

Pittsburgh Central Catholic High SchoolGrade 9

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?

Problem

4 main ingredients: Ethanol, Difluoroethane, Polyaminopropyl biguanide, and a secret unknown fragrance substance

Popular among male teens

Axe Aerosol Body Spray

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.

Microbial Skin Flora

Gram-positive Part of the normal skin flora Grows best in aerobic conditions even though

it is anaerobic Form clusters and can create biofilms

Staphylococcus epidermidis

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

Purpose

Null Hypothesis: Axe aerosol body spray will have no effect on bacterial survivorship

Alternative Hypothesis: Axe aerosol body spray will significantly reduce bacterial survivorship

Hypothesis

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

Materials:

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%

Procedures: Expt. 1 (Liquid Pulse)

Tube 110%

Tube 21%

Tube 30.1%

Tube 40%

Sterile dilution fluid

8.9 mL 8.9 mL 8.9 mL 8.9 mL

Microbe 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Sterile fluid 0 mL 0.9 mL 0.99 mL 1.0 mL

Axe body spray

1.0 mL 0.1 mL 0.01 mL 0 mL

Total Volume

10 mL 10 mL 10 mL 10 mL

Table 1: Chart of Concentration (Expt. 1)

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 103 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.

Procedures:

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

ANOVA and Dunnett’s Test

0 0.1 1 100

100

200

300

400

500

600

700

601545

108

0

[Axe Body Spray %]

Colo

nie

sEffect of Axe body spray on Staph(Liquid Pulse)

P-Value=1.3729E-13

Dunnett’s Test: Expt. 1

T-Critical = 2.54Alpha= 0.05

Aerosol Body Spray

Concentration T-Value Interpretation

0.1% 1.54 Not significant

1% 13.58 Significant

10% 16.55 Significant

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.

Procedure: Agar Infusion

Effect of Axe body spray on Staph (Agar Infusion)

0 uL 2 uL 20 uL 200 uL0

100

200

300

400

500

600

700601 581.75 564.25

148.5

Axe body spray volume

Resu

ltin

g n

um

ber

of

colo

nie

s

P-Value= 2.02E-07

Dunnett’s Test: Expt. 2

T-Critical = 2.63Alpha= 0.05

Aerosol Body Spray

Amount T-Value Interpretation

2 uL 0.43 Not significant

20 uL 0.83 Not significant

200 uL 10.18 Significant

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.

Results Summary

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.

Conclusions/Recommendations

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

Limitations and Extensions

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.elephantjournal.com/2010/03/you

r-little-man-on-axe-elizabeth-joseph/ http://www.natural-skincare-authority.com/ax

e-deodorant-body-spray.html#top http://www.allure.com/beauty-trends/blogs/da

ily-beauty-reporter/2013/03/axe-body-spray-life-threatening-allergic-reaction.html

http://www.textbookofbacteriology.net/themicrobialworld/NormalFlora.html

Sources

Number of Colonies Per PlatePlate 1

Plate 2

Plate 3

Plate 4

Plate 5

Plate 6

Mean

0% 562 755 617 552 621 501 601

0.1% 490 505 550 632 563 527 545

1% 50 37 189 35 164 175 108

10% 0 0 0 0 0 0 0

Data Results (Expt. 1)

Number of Colonies Per PlatePlate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Mean

0 mL† 562 755 617 552 621 501 601

2 mL 585 463 642 637 n/a n/a 582

20 mL 552 564 581 560 n/a n/a 564

200 mL

182 118 129 165 n/a n/a 149

Data Results (Expt. 2)

† Data from Experiment 1

n/a = not applicable

ANOVA: Expt. 1ANOVA: Single Factor

SUMMARYGroup (concentration) Trials Sum Average Variance0% 6 0 0 00.10% 6 650 108.3333 5583.8671% 6 3267 544.5 2573.110% 6 3608 601.3333 7658.667

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 1659508 3 553169.3 139.9044 1.37294E-13 3.098391Within Groups 79078.17 20 3953.908

Total 1738586 23

ANOVA: Expt. 2ANOVA: Single Factor

SUMMARYGroups Count Sum Average Variance

0 mL 6 3608 601.3333 7658.6672 mL 4 2327 581.75 6931.58320 mL 4 2257 564.25 149.5833200 mL 4 594 148.5 901.6667

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 596519.9 3 198840 44.7249 2.02E-07 3.343889

Within Groups 62241.83 14 4445.845

Total 658761.8 17