1. EFFECTS OF FRACKING FLUID ON STAPH. EPIDERMIDIS AND E. COLI LUKE WEARDEN GRADE 11 CENTRAL CATHOLIC HIGH SCHOOL

2. PROBLEM How do fracking chemicals influence the survivorship of Escherichia coli and Staphylococcus epidermidis?

3. FRACKING BACKGROUND Use: accesses natural gas found in subterranean shale Fracking is effective in obtaining energy, but can be harmful to the environment Many environmentalists groups including the EPA have taken strong anti-fracking stances Are living organisms at risk from fracking?

4. HOW FRACKING WORKS

5. FRACKING SOLUTION Chemical Sodium Chloride Calcium Chloride Magnesium Chloride Potassium Chloride Barium Chloride Strontium Chloride Amount (mg/L) 75.6516.650.4750.38460.18570.20

6. GRAM (-) VS. GRAM (+) BACTERIA Cell wall 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.

7. STAPHYLOCOCCUS EPIDERMIDIS (STAPH) Common surface symbiont in many mammals (Human). Gram (+) bacteria Most strains considered non-pathogenic. Pathogenic strains can be life-threatening.

8. ESCHERICHIA COLI (E.COLI) Large and diverse group of gram (-) bacteria Free living, symbionts, or pathogens Live in the intestinal tract of many mammals. Most strains are not pathogenic Serve as a common prokaryotic cell model.

9. PURPOSE Assess the effects of fracking chemicals on the survivorship of Staphylococcus epidermidis and Escherichia coli.

10. HYPOTHESES Null Hypothesis: There will be no significant effect of fracking chemicals on the survivorship of E.coli and Staph. Alternative Hypothesis: The fracking chemicals will significantly reduce the survivorship of the bacteria.

11. MATERIALS Fracking Chemicals Micro and Macro pipettes + Sterile tips Spreader bars LB agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl) Escherichia coli DH5-alpha(E.Coli) Staphylococcus epidermidis Burner Sidearm Flask Vortex Incubator Gloves/goggles SDF (Sterile Dilution Fluid 10 mm KH2PO4, 1 mm MgSO4, 0.1 mm CaCl2, 100 mm NaCl) 20 Sterile Test Tubes Ethanol Luria Broth Agar Plates(To be Infused with Agar Plates) 0.22 micron syringe filters and 10mL syringe Klett Spectrophotometer

12. PROCEDURE 1.Bacteria (E.coli and Staph) were grown overnight in sterile LB Media. 2.Samples of the overnight cultures 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. 4.The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 ⁵ cells/mL. 5.The fracking chemicals were sterilized by means of a 0.2 micron syringe filter 6.The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 0.01%, 0.1%, 1%.

13. CONCENTRATION CHART Ingredients0%0.01%0.1%1% Sterile Fluid added 1mL0.999mL0.99mL0.9mL E.coli/Stap h 0.1mL Fracking Chemicals 0mL0.001mL0.01mL0.1mL Original Sterile Fluid 8.9mL Total Volume 10mL

14. PROCEDURE 7.100 µL of cell culture was then added to the fracking chemical solutions, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 8.The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 9.After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB-agar plates. 10.The plates were incubated at 37°C for 24 hours. 11.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

15. ANOVA AND DUNNETT’S TEST ANOVA Statistical test that allows the comparison of means of different groups Dunnett’s Test Comparison of experimental and control group to determine significant variation

16. DATA P-value: 5.472E-28 P-value: 2.994E-28

17. DUNNETT'S TEST – E. COLI ConcentrationT-valueSignificance 0.01%2.46Significant 0.1%3.77Significant 1%4.57Significant T-crit = 2.365

18. DUNNETT’S TEST - STAPH ConcentrationT- ValueSignificance 0.01%2.69Significant 0.1%3.77Significant 1%4.58Significant T-crit =2.365

19. CONCLUSIONS Reject the null hypothesis that fracking chemical will not have a significant effect of Staph and E. coli survivorship. At every concentration for both bacteria the fracking fluid significantly reduced survivorship. Accept the alternate hypothesis that fracking fluid will have a significant effect on Staph and E. coli survivorship. An increase in the concentration of fracking fluid will generally correlate with a significant decrease in the survivorship of Staph and E. coli.

20. LIMITATIONS Plating was slightly unsynchronized Only 4 concentrations were tested Only one exposure time was used Only one method of exposure used

21. FUTURE STUDIES Use a wider variety of concentrations of fracking fluid Vary exposure times Use various cell models Test for synergistic effects

22. REFERENCES https://fracfocus.org/chemical-use/what-chemicals-are-used http://www.huffingtonpost.com/news/fracking-chemicals/ https://etd.ohiolink.edu/ap/10?0::NO:10:P10_ACCESSION_NUM:osu1366292190 http://onlinelibrary.wiley.com/doi/10.1002/etc.2619/abstract?deniedAccessCustomised Message=&userIsAuthenticated=false http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/ http://www.webmd.com/a-to-z-guides/e-coli-infection-topic-overview