1. Body Ink Effects on Microbial Survivorship Kevin Frankola Pittsburgh Central Catholic Highschool Grade 11

2. Tattoo Ink 24% of Americans between the ages of 18- 50 have a tattoo One of the more common types of tattoo ink was selected, an ink containing an Azo Compound. Azo Compounds are characterized by the functional group: R-N=N-R‘ R and R' can be either aryl or alkyl.

3. Tattoos Tattoo ink is injected underneath epidermis Tattoo ink permanently stains dermis The dermis contains sweat glands, sebaceous glands, and some sensory cells The ink could possibly interfere with the role of these cells.

4. Escherichia coli Most researched prokaryotic cell Gram negative Found in the intestines of warm-blooded animals Prokaryotic cells lack a nucleus, their genetic code wrapped in a bundle in the center of the cell Strong enough chemicals can alter DNA causing potentially fatal mutations

5. Yeast The most researched cell Eukaryotic cell Fungi cell Dominate ocean fungal diversity Usually 3–4 µm in diameter Have the stronger relationship to Human Skin cells, both being Eukaryotic

6. Purpose Humans absorb many chemicals Can chemicals that come into contact with the skin have harmful effects? The purpose of this experiment was to determine if tattoo ink has a significant effect on microbial growth. Prokaryotic cells and eukaryotic cells were used, Escherichia coli and Yeast respectively

7. Hypotheses The alternative hypothesis is that any variation in survivorship will not be due to chance, but rather the ink has an effect on microbial survivorship. The null hypothesis is that the ink will have no effect on microbial survivorship and that any variation in survivorship is due to chance alone.

8. Materials Culture of yeast cells at 10 ⁵ cells per ml. Culture of Escherichia coli at 10 ⁵ cells per ml. 164.56ml of SDF 1ml of tattoo ink 16 tubes 32 YEPD agar plates (yeast) 32 Nutrient agar plates (E. coli) Micro pipettes (1000μl and 200 μl) Burner Vortex Spreader Bar Ethyl alcohol Sterile tips

9. Procedure 1. Saccharomyces cerevisiae was grown overnight in sterilized YEPD media. 2. A sample of the overnight cultures was added to fresh media in a sterile sidearm flask. 3. The culture of yeast was incubated at 30°C until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells per mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells per mL. The following tubes were created: 5. The tubes were incubated at room temperature for 30 minutes. 0.1 mL of each suspension was then plated onto YEPD agar plates. The plates were incubated at 30C for 48 hours. The resulting colonies were counted (each colony was assumed to have arisen from one cell.

10. Procedure (Continued) 6. 100μl of yeast culture were pipetted into each of the 8 yeast-designated tubes. 7. 100μl of Ecoli culture were pipetted into each of the 8 Ecoli- designated tubes. 8. 100μl of a tube’s solution was pipetted onto a plate. 9. The spreader bar was sterilized, then used to evenly spread solution over the plate. 10. Steps 8-9 were repeated for every tube 4 times, totaling of 32 plates of Ecoli cultures and 32 plates of yeast cultures. 11. The plates were incubated for 36 hours. 12. The colonies on the plates were counted the following day.

11. Yeast

12. E. coli

13. P value = 0.311468

14. P value = 0.078993

17. Conclusion The results appear to show the ink had no significant effect on the survivorship of yeast and E. coli. All four of the ANOVA P values indicated that the null hypothesis must be accepted. The ink had no effect on microbial survivorship and any variation in survivorship was due to chance alone.

18. Limitations and Extensions 10% concentration would be highly increased Different colors would be tested Different species Test on macro organisms

19. References www.mayoclinic.com www.wikipedia.org www.fda.gov www.chemistry.about.com www.tattooology.com