1. Does Estrogen Affect Escherichia coli Survivorship? Robert McKinstry Central Catholic High School

2. Aquatic Pollution Problems Run-off: Debris Dirt Chemicals Sewage and other components Cleaning chemicals Pharmaceuticals

3. Pharmaceutical Contamination Recent India Study Drugs: ciprofloxacin, treatments for hypertension, heart disease, chronic liver ailments, depression, gonorrhea, ulcers and other ailments

4. Estrogen Chemical steroid. Functions as the primary female sex hormone. Promotes the growth of secondary female sex characteristics, accelerates metabolism, reduces muscle mass, and decelerates height growth.

5. Estrogen As a Pharmaceutical Treatments: Osteoporosis Symptoms of menopause Used in hormone replacement therapy and birth control pills.

6. How Estrogen Enters Water a) Flushed down drains in the form of urine b) The discarding of estrogen pills c) Accidently dumped by industries

7. Estrogen Contamination High levels – Improper disposal Near-impossible to remove Florida Museum: “When adult male fish are exposed to short term and low concentrations of a synthetic estrogen, their fertility can drop by as much as 50%, according to a study by scientists at the Department of Energy's Pacific Northwest National Laboratory (PNL).”

8. Escherichia coli Large and diverse group of gram (-) bacteria Free living, symbionts, or pathogens U.S. infections > 70,000 per year Large outbreaks rare, sporadic food infestations Serves as a common prokaryotic cell model.

9. E. coli and Estrogen E. coli and other coliforms are common in rivers across the world. Estrogen is a growing problem in many of the same rivers and tributaries. Does estrogen affect E. coli populations?

10. Purpose To determine if estrogen has an affect on E. coli survivorship in vitro.

11. Hypotheses Null hypothesis: Estrogen does not affect E. coli survivorship in vitro. Alternative hypothesis: Estrogen does affect E. coli survivorship in vitro.

12. Labeling tape Ethanol Latex gloves E. coli DH5 Alpha Micropipettes Micro rack Ten micro tubes LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride). Mortar and Pestle Materials Estrogen (0.5 mg pills) Sterile Dilution Fluid (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl) Turntable 40 Luria Broth agar plates Bunsen burner Spread bar Incubator Klett Spectrophotometer Matches

13. Procedure 1. E. coli 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 10 8 cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 5. An estrogen suspension was mixed with the appropriate amount of SDF to create estrogen concentrations of 1%, 0.1%, and 0.01%.

14. Procedure 6. 100 µL of cell culture was then added to the estrogen solutions, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 8. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB plates. 9. The plates were incubated at 37 C for 24 hours. 10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

15. Chart of Concentration 0% Concentration 0.01% Concentration 0.1% Concentration 1% Concentration Microbe0.1mL SDF9.9mL9.8999.89mL9.8mL Estrogen Solution 0mL0.001mL0.01mL0.1mL Total10mL

16. 10 2 cells 10 3 cells/mL 10 5 cells/mL with estrogen 10 8 cells/mL (E. coli) 100 uL 10 5 cells/mL (E. coli) 100 uL

17. P >.05 P <.05 Colony Count 0%0.01%0.1%1% P-value 2.59E-08

18. Variable Concentration T valueInterpretation 0.01% Estrogen1.50Not Significant 0.1% Estrogen1.19Not Significant 1% Estrogen6.19Significant Dunnett’s Test Analysis T Critical = 2.76 (significant) Alpha =.05

19. Conclusions The null hypothesis that Estrogen does not affect E. coli survivorship in vitro was rejected for the concentration of 1% estrogen. The null hypothesis could not be rejected for the lower concentrations of 0.01% and 0.1% estrogen. The results indicate that 1% estrogen did negatively influence E. coli survivorship.

20. Limitations, Extensions, and Future Studies The E. coli plating was not exactly synchronized, possibly contributing to varying colony counts. The estrogen was quite insoluble, requiring the use of a suspension. Larger sample sizes will be employed in the next round of testing. Higher and lower concentrations of estrogen will be tested. Directly infusing estrogen into the LB agar for prolonged cell exposure. Limitations Extensions and Future Studies

21. References Estrogen Makes Fish Too Feminine To Reproduce http://www.flmnh.ufl.edu/fish/InNews/estrogen2007.html Short Term Exposure to Estrogen Cuts Fish Fertility http://www.flmnh.ufl.edu/fish/InNews/fishfert2003.htm Estrogen knocks out fish in whole-lake experiment http://pubs.acs.org/subscribe/journals/esthag- w/2003/aug/science/jp_lake.html http://www.ninemilerun.org/resources/scientific_reports/Bacteria%20&%20D etergent%20Results%20August%202006.pdf http://www.nmenv.state.nm.us/swqb/wps/San_Juan/SanJuanRiver- 2004BacteriaSamplingReport.pdf http://www.cdc.gov/nczved/dfbmd/disease_listing/stec_gi.html

22. Results 0% Estrogen0.01% Estrogen0.1% Estrogen1% Estrogen 56801044 86807018 8694544 761047038 86985244 10092726 689474100 86946828 70907032 74806028 Colony Counts

23. P <.05 P >.05 0%0.01%0.1%1%