1. Triclosan’s Effect on Yeast Survivorship Ryan McNelis Freshman, Pittsburgh Central Catholic

2. Triclosan Acts against bacteria and fungi in a variety of commercial products including, soaps, shampoos, and toothpastes Works by targeting cytoplasmic and membrane targets Has been reported to have various negative health effects, ranging from increasing risks of food allergies, to muscle contraction. It has also been found toxic to bacteria at levels that are found throughout aquatic environments.

3. Saccharomyces cerevisiae(Yeast) Saccharomyces cerevisiae (Yeast) Eukaryotic microorganism Unicellular 3–4 µm diameter The most studied cellular model in research Cell cycle is similar to human cells Comparable DNA replication, recombination, cell division and metabolism

4. Purpose The purpose of this experiment was to determine the effect varying concentrations of triclosan had on the survivorship of yeast cultures

5. Hypothesis Null- triclosan will have no significant effect on the number of surviving yeast colonies Alternative- triclosan will significantly decrease the number of surviving yeast colonies

6. Materials Test tubes Proper safety equipment Water Spreader bars Vortex Sterile Water Hot Plates Beakers Test tube rack Micropipettes Pipette tips Yeast (Saccharomyces cerevisiae) YEPD Agar Plates YEPD Media (0.5% yeast, 2% Glucose, 2% Peptone) Triclosan Sterile Dilution Fluid

7. Procedure (Pulse Exposure) 1. S.c. yeast was grown overnight in sterile YEPD media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in a incubator (30 C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/mL. 4. The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/mL. 5. A stock of 0.3% triclosan was created (hereafter x) 6. Using this stock of x solutions of the following concentrations were created; x, 0.1x, 0.01x, 0.001x, 0.0001x, 0.00001x, and 0x. 0.1ml of yeast was pipetted into a 9.9ml volume of each of these solutions 7. 0.1 mL of the yeast/variable concentration was pipetted onto five plates per variable. 8. The plates were incubated at 30 C for 48 hours. 9. The resulting colonies were counted. Each colony is assumed to have risen from one cell.

8. Procedure (Continuous Contact Exposure) 1. S.c. yeast was grown overnight in sterile YEPD media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in a incubator (30 C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/mL. 4. 0.1 mL of each variable at concentration of 1X (recommended dosage) was pipetted onto six plates. 5. 0.1mL of a substock of x, with a concentration of.001x, was pipetted onto six plates. 5. The plates were labeled and the agar was allowed to absorb the variable for thirty minutes. 6. The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/mL. 7. After vortexing to evenly suspend cells, 0.1mL aliquots were removed from the tubes and spread on YEPD plates. 8. The plates were incubated at 30 C for 48 hours. 9. The resulting colonies were counted. Each colony is assumed to have risen from one cell.

9. Pulse Experiment Surviving Colonies Concentration (x =.3% Triclosan)

10. Pulse Experiment (Survivorship Curve)

11. Infused Results

12. Statistical Analysis Pulse Experiment ConcetrationT ValueCompared to T crit (3.73) 0.00001x3.20NOT SIGNIFIGANT 0.0001x5.27SIGNIFIGANT 0.001x2.03NOT SIGNIFIGANT 0.01x1.02NOT SIGNIFIGANT 0.1x11.80SIGNIFIGANT x17.31SIGNIFIGANT

13. Statistical Analysis Infused ConcetrationT ValueCompared to T crit (3.03) Low0.53NOT SIGNIFIGANT High3.24SIGNIFIGANT

14. Conclusion The null hypothesis can be rejected. For both experiments, triclosan concentration had a generally negative correlation with the number of surviving colonies

15. Limitations A serious limitation in this experiment was that the powdered triclosan was unable to completely dissolve in the dilution that was created. As a result, the amount of triclosan in the various dilutions may have been slightly skewed because the mixture was not completely homogeneous.

16. Continuations Testing the effects of triclosan on other micro organisms, such as bacteria. Testing environmentally friendly alternatives to triclosan as an anti microbial agent, such as hydrogen peroxide.

17. Works Cited http://www.fda.gov/ForConsumers/Consumer Updates/ucm205999.htm http://www.fda.gov/ForConsumers/Consumer Updates/ucm205999.htm http://www.epa.gov/oppsrrd1/REDs/factsheet s/triclosan_fs.htm http://www.epa.gov/oppsrrd1/REDs/factsheet s/triclosan_fs.htm http://en.wikipedia.org/wiki/Triclosan