1. The Effects of Acne Medication on Bacteria Survivorship By: Maria DeRenzo Grade: 9 Oakland Catholic High School

2. Bacteria Prokaryotic microorganisms Often the cause of human and animal disease Found everywhere 4 Basic shapes 1.Cocci (round) 2.Bacilli (rods) 3.Vibrios (bent rod) 4.Spirilla (spirals)

3. Escherichia coli (E. coli) One of the most common forms of bacteria found in many environments Rod-shaped Gram negative bacterium Part of the human flora; found in the human colon and digestive tract Reproduces rapidly, often within thirty minutes Most are non-pathogenic

4. Staphylococcus Epidermidis Common surface symbiont in many mammals (humans) Gram positive Most forms are considered non-pathogenic Part of human skin flora Forms biofilms

5. Gram (-) vs. Gram (+) Bacteria Cell was contains an extra layer of lipopolysaccharides for extra protection. Outer membrane protects bacteria from several antibiotics. Most pathogenic bacteria in humans are Gram (+) organisms. Simple cell wall. Some antibiotics work against the formation of the cell wall.

6. Acne Common human skin disease Areas of skin with seborrhea (scaly red skin), comedones (blackheads and whiteheads), pimples, and possible scarring Excess amount of sebum and dead skin cells: bacteria can thrive Caused by hormones, diet, stress, medications, and genetic factors Treated by over-the-counter and prescription medications in the form of a pill or wash

7. Acne Medication Clean & Clear Deep Cleaning Astringent Oil-free Used for the treatment of acne To stop and prevent pimples Active Ingredients: Salicylic Acid - 2% Acne Medication Inactive Ingredients: Alcohol, Benzophenone-4, Denatonium Benzoate, Dimethicone Propyl PG-Betaine, Fragrance, Isoceteth-20, Water, Red 4

8. Key Question and Hypotheses Question: What is the Effect of Acne Medication on Bacteria Survivorship? Null Hypothesis: Acne medication will not significantly reduce survivorship of both E. coli and Staphylococcus epidermidis. Hypothesis: Acne medication will significantly reduce survivorship of both E. coli and Staphylococcus epidermidis.

9. Materials E. coli bacteria Staphylococcus epidermidis bacteria Clean & Clear Deep Cleaning Astringent Oil-free Micro and macro pipettes and tips Bacterial spreader, ethanol, burner, turn table 92 LB agar plates Beaker Tube racks Vortex Incubator Sterile tubes + sterile dilution fluid (SDF) Klett Spectrophotometer

10. Variables The independent variable in this experiment was Clean & Clear Deep Cleaning Astringent Oil- free The dependent variable in this experiment was the survivorship of the e. coli and Staphyloccous Epidermidis

11. Procedure A. Liquid Exposure 1. Bacteria (E. coli and Staph e.) were grown overnight in sterile LB media. 2. Samples of the overnight culture for E. coli and Staph were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8 cells/mL. The cultures were then diluted in SDF to approximately 10^5 cells/mL. 4. The Clean and Clear Deep Cleaning Astringent was diluted with sterile dilution fluid in test tubes into concentrations of 1%, 10%, and 50% with a fourth tube containing 0% of the acne medicine to be used as a control. 5. The microbe was then added to the test tubes to yield a total of 10 mL in each test tube. 6. The tubes were allowed to sit for 5 minutes; 100uL aliquots were then spread onto LB agar plates. 7. Plates were incubated at 37C o/n and resulting colonies counted.

12. Chart of Concentrations (mL) Liquid Exposure 0% Stock (Control) 1% Stock10% Stock50% Stock Microbe.1 mL SDF9.9 mL9.8 mL8.9 mL4.9 mL Clean & Clear 0 mL.1 mL1 mL5 mL Total10 mL

13. Procedure cont. B. Solid Agar Infusion Procedure 1. The medication was infused into the agar by spreading 100 or 200 uL of stock onto the agar. Plates were inverted and incubated at 37C for 2 hours to allow infusion. 2. 100uL of CONTROL cell suspension was spread onto the infused plates. 3. The plates were incubated overnight and resulting colonies counted. C. Liquid Agar Infusion Procedure 1. Medication was infused directly into liquid agar at concentrations of 1% and 10%. 2. 100uL of CONTROL cell suspension was spread onto the infused plates. 3. The plates were incubated overnight and resulting colonies counted.

14. Chart of Concentrations (mL) Infusion 0% Acne Medicine 1% Acne Medicine 10% Acne Medicine LB Growth Media 249.9 mL247.4 mL224.9 mL Clean & Clear0 mL2.5 mL25 mL Microbe.1 mL Total250 mL

15. Liquid Agar Infusion Medicine Effects on E. coli Number of Colonies

16. Liquid Agar Infusion Medicine Effects on Staph e. Number of Colonies P- Value = 1.84E-09

17. Effects of Acne Medicine on E. coli (Liquid Exposure) Number of Colonies P- Value = 2.39E-16

18. Effects of Acne Medicine on Staph e. (Liquid Exposure) Number of Colonies [Clean & Clear Deep Cleaning Astringent] P- Value = 2E-21

19. Solid Agar Infusion Medicine Effects on E. coli Number of Colonies [Clean & Clear Deep Cleaning Astringent] P- Value = 1.3E-07

20. Solid Agar Infusion Medicine Effects on Staph e. [Clean & Clear Deep Cleaning Astringent] Number of Colonies P- value = 3.33E-09

21. Anova: Single Factor Analysis of Variation Statistical test that asses the differences between means (averages) Used when the experiment involves three or more levels of a single independent variable Tests hypotheses about the average of a dependent variable across different groups Determines whether or not null hypothesis can be rejected or accepted

22. Anova: Single Factor E. coli Factors being testedP- ValueInterpretation (Infusion) (0%) (1%)3.96E-06Significant (Liquid Exposure) (0%) (1%) 2.39E-16Significant (Spread) (0 mL) (0.1 mL) (0.2 mL) 1.3E-07Significant Alpha = 0.01

23. Anova: Single Factor Staph Factor being testedP- ValueInterpretation (Infusion) (0%) (1%)1.84E-09Significant (Liquid Exposure) (0%) (1%) 2.39E-16Significant (Spread) (0 mL) (0.1 mL) (0.2 mL) 3.33E-09Significant Alpha = 0.01

24. Dunnett’s Test Quantitative statistical analysis that compares means (averages) All variation groups are compared to reference group (control) Identifies variation groups with means that significantly differ from the reference group

25. Dunnett’s Test E. coli Variable ConcentrationT ValueInterpretation (Infusion) 1%7.05032701126Significant (Liquid Exposure) 1%3.82747085938Significant (Spread) 0.1 mL7.28216111454Significant (Spread) 0.2 mL12.452926403Significant

26. Dunnett’s Test Staph e. Variable ConcentrationT ValueInterpretation (Infusion) 1%10.2740720413Significant (Liquid Exposure) 1%11.3710213075Significant (Spread) 0.1 mL14.002664108Significant (Spread) 0.2 mL15.7689299318Significant

27. Conclusions Null Hypothesis: Acne medication will not have a significant effect on the growth of either e. coli or Staphylococcus Epidermidis. Rejected Hypothesis: Acne medication will have a significant effect on the growth of either e. coli or Staphylococcus Epidermidis. Accepted

28. Limitations Extensions There was a lag time when plating the cells. Exposure times to the Clean and Clear Deep Cleaning Oil- Free Astringent varied slightly. Test greater variety concentrations of variable Use different bacteria

29. References http://www.mayoclinic.com/health/acne/DS00169/DSECTION =causes] http://www.mayoclinic.com/health/acne/DS00169/DSECTION =causes http://en.wikipedia.org/wiki/Sebaceous_gland http://en.wikipedia.org/wiki/Acne_vulgaris http://www.sciencebuddies.org/science-fair- projects/project_ideas/MicroBio_p019.shtml http://www.sciencebuddies.org/science-fair- projects/project_ideas/MicroBio_p019.shtml http://www.mayoclinic.com/health/e-coli/DS01007 http://www.medicalnewstoday.com/articles/68511.php http://web.uconn.edu/mcbstaff/graf/Student%20presentation s/S%20epidermidis/sepidermidis.html http://web.uconn.edu/mcbstaff/graf/Student%20presentation s/S%20epidermidis/sepidermidis.html http://en.wikipedia.org/wiki/Gram-negative_bacteria