1. Acne Medication Antimicrobial Effects
Fiorente Pampena
Pittsburgh Central Catholic High School
Grade 9

2. Acne Vulgaris Common human skin disease
Caused by bacteria, hormones, diet, steroids
Bacteria thrives with excess sebum and dead skin cells
Whiteheads, blackheads, nodules, pustules, papules
Treated by over-the-counter and prescription medicines
Pills, washes, creams

3. Clearasil Stubborn Acne Control Pads
Cotton pads soaked in medication
Active ingredient is salicylic acid
Salicylic acid helps correct abnormal shedding of cells
Cleans outer layer of skin by unclogging pores and preventing lesions
1.9% salicylic acid by volume

4. Witch Hazel Hamamelis, genus of deciduous shrubs and trees
Medical astringent
Cuts, scrapes, bug bites, acne
Constricts body tissue
Minimizes pores and dries oil
Contains tannins, which have antioxidant properties
Medication used 86% by volume

5. Human Microbial Flora
The microorganisms inside or on the surface of the human body
Includes bacteria, fungi, and archaea
Can cause disease if growing in uncommon areas or large numbers
An estimated 10% of the body mass is symbionts
Do acne medications interfere with human microflora?

6. Gram (-) vs. Gram (+) Bacteria
Gram-Negative
Gram-Positive
Single layer of peptidoglycan
Cell wall contains extra layer of lipopolysaccharides
For outer protection
Protects bacteria from antibiotics
Several layers of peptidoglycan
Antibiotics work against formation of cell wall
Most pathogenic bacteria in humans are gram-positive

7. Cutibacterium acnes Gram-positive Part of human microflora
Live deep within follicles and pores
Attributed to acne vulgaris
Secrete digestive enzymes
Destabilize the cells that form the walls of the follicle
Rapid growth caused by cellular damage in follicles triggers inflammation

8. Staphylococcus epidermidis
Common surface symbiont in many mammals
Gram-positive
Most forms considered non-pathogenic
Part of human microflora
Attributed to acne vulgaris along with C. acnes
Used as the microflora model in this experiment

9. Alternative Hypothesis
Hypotheses
Null Hypothesis
Alternative Hypothesis
There will be no difference in the survivorship of Staphylococcus epidermidis bacteria in the plates containing witch hazel or Clearasil.
There will be a difference in the survivorship of Staphylococcus epidermidis bacteria in the plates containing witch hazel or Clearasil.

10. Materials Staphylococcus epidermidis bacteria
Clearasil Stubborn Acne Control Pads
Witch hazel
Sterile micro/macro pipettes and tips
Bacterial spreader, ethanol, burner, turn table
LB agar plates (1% Tryptone, 0.5% Yeast Extract, 1% NaCl)
Beaker
Test tubes
Test tube racks
Vortex
Incubator (37°C)
Sterile dilution fluid (SDF)
Sterile disks and forceps
Sterile water

11. Purpose
To determine which of the two acne medications (witch hazel and Clearasil) work best at killing and preventing bacteria when applied to human skin.

12. Procedure A. Liquid Exposure
1. Bacteria (S. epidermidis) were grown overnight in sterile LB media.
2. Sample of the overnight culture of S. epidermidis were 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. The culture was then diluted in SDF to approximately 10⁵ cells/mL.
4. Medications were diluted with SDF in test tubes into two concentrations with a third tube containing 0% of the medications to be used as a control.
5. The microbe was then added to the test tubes to yield a total volume of 10 mL in each tube.
6. The tubes were allowed to sit for 5 minutes; 100μl aliquots were then spread onto LB agar plates.
7. Plates were incubated at 37°C overnight and resulting colonies counted.

13. Concentrations (mL) of Liquid Pulse Exposure
0% Stock
(Control)
0.1% Stock
1% Stock
Microbe
(S. epidermidis)
0.1 mL
Sterile Dilution Fluid
(SDF)
9.9 mL
8.9 mL
7.9 mL
Acne Medication
0 mL
1 mL
2 mL
Total
10mL

14. Procedure cont. B. Agar Infusion
Transferred 200μl of medications and sterile water onto LB agar plates.
Transferred 180μl of sterile water and 20μl of medications onto other plates.
Spread liquid evenly on every plate, getting a new sterile spreader for each plate.
Incubated plates in a 37°C incubator until solutions were absorbed into agar.
Spread S. epidermidis evenly on every plate, getting a new sterile spreader for each plate.
Plates were incubated at 37°C overnight and resulting colonies counted.

15. Procedure cont. C. Zones of Inhibition
Spread S. epidermis evenly on LB agar plates with a sterile spreader and incubated in a 37°C incubator.
Dipped sterile disk in acne medications (100% concentration) using sterile forceps.
Placed disks with medications around perimeter of plate. (4 disks on each plate)
Dipped sterile disk in a dilution of medications (10% concentration) using sterile forceps.
Placed disks with 10% dilution of medications around perimeter of plate.
Plates were incubated at 37°C overnight and resulting colonies counted.

16. Acne Medication Effects on Staph Survivorship (Liquid Pulse Exposure)
P-value: 3.09E-22

17. Liquid Pulse Dunnett's Test
Acne Medication Concentrations
T-value
Conclusion
Clearasil 1%
47.44
Significant
Clearasil 10%
47.66
Witch Hazel 1%
13.96
Significant 
Witch Hazel 10%
19.52

18. Acne Medication Effects on Staph Survivorship (Agar Infusion)
P-value: E-22

19. Agar Infusion Dunnett's Test
Acne Medication
T-value
Conclusion
Clearasil 200μl
34.26
Significant
Clerasil 20μl + 180μl Sterile Water
Witch Hazel 200μl
 19.22
Witch Hazel 20μl + 180μl Sterile Water
14.11

20. Acne Medication Effects on Staph Survivorship (Zones of Inhibition)
P-value: E-15

21. Zones of Inhibition Dunnett's Test
Acne Medication Concentrations
T-value
Conclusion
Clearasil 100%
37.88
Significant
Clearasil 10%
29.97
Witch Hazel 100%
14.26
Witch Hazel 10%
10.7

22. Conclusion
All concentrations of both acne medications significantly reduced survivorship of Staphylococcus epidermidis bacteria.
Null hypothesis is rejected because the Clearasil acne medication had significantly higher T-values than the witch hazel in all Dunnett's tests.

23. Limitations There was lag time when plating the microbe
Exposure times to the medications varied slightly
Study does not reveal other effects on bacterial health
Disks were left opened for a short period of time
Only addresses survivorship
Limited range of exposures
Limited exposure time
Only 1 species used

24. Extensions More trials Different microbes
Different concentrations of medication
Medications used together in different combinations to determine if any synergistic effects
Determine if the medications effect the growth rate of the bacteria in addition to survivorship

25. References
AR, Shalita. “Department of Dermatology, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA. & Health, StatPearls Publishing, europepmc.org/abstract/med/
Toombs, Ella. “Cosmetics in the Treatment of Acne Vulgaris.” NeuroImage, Academic Press, 21 July 2005,
Otto, Michael. “Staphylococcus Epidermidis - the 'Accidental' Pathogen.” Nature News, Nature Publishing Group, 1 Aug ,
Reuter, Juliane, et al. “Which Plant for Which Skin Disease? Part 1: Atopic Dermatitis, Psoriasis, Acne, Condyloma and Herpes Simplex.” The Canadian Journal of Chemical Engineering, Wiley-Blackwell, 5 Aug. 2010, onlinelibrary.wiley.com
Scalbert, Augustin. “Antimicrobial Properties of Tannins.” NeuroImage, Academic Press, 15 May 2001,
Gollnick, H., and M. Schramm. “Topical Drug Treatment in Acne.” Dermatopathology, Karger Publishers, 13 Mar. 1998,

26. Statistical Analyses ANOVA: Single Factor Dunnett's Test
Analysis of Variation
Statistical test that compares the means of multiple groups
P-value from ANOVA testing identifies significance if < 0.05
Statistical test that compares experimental group directly to control group
Identifies that experimental group is significantly different than the control if t-stat > t-crit

27. Liquid Pulse ANOVA

28. Agar Infusion ANOVA

29. Zones of Inhibition ANOVA