1. Tyler Barkich Grade 9 Pittsburgh Central Catholic High School

2.  Any chemical, antibacterial, biological agent, or other similar substance used to kill or repel unwanted species.  Used commonly in agriculture and around homes  877 million pounds of pesticides were used agriculturally in 2007.  Bayer Pesticide Active Ingredients:β-cyfluthrin and imidacloprid.

3.  Research suggests over 100 active pesticide ingredients can cause cancer or birth defects.  Pesticides have been linked to endocrine disruption.  Pyrethroids, a type of ingredient found in the pesticide used in this experiment, are toxic to most forms of life.  An estimated 98% of sprayed insecticide and 95% of herbicide are not used on target species.

4.  Rod shaped cells, usually 2 nanometers in length  Prokaryotic  Gram-negative  Found in the intestines of many mammals  Commonly used model  Reproduces rapidly, usually within thirty minutes  Many strains, most non-pathogenic

5.  Bacteria that is mostly harmless and lives normally on skin and mucous membranes of humans  Gram-positive  Many forms are considered non-pathogenic  Pathogenic forms can be lethal

6. Gram+ Most pathogenic Simple cell wall. Antibiotics work against the formation of the cell wall. Staphylococcus epidermidis Gram- The cell wall is a thin layer of lipopolysaccharide, which adds extra protection. This layer protects the cell from certain antibiotics. Escherichia coli

7.  Do pesticides have an effect on the microbial populations in natural ecosystems?

8.  To determine if the Bayer Advanced Pesticide in different concentrations will affect the survivorship or Escherichia coli or Staphylococcus epidermidis.

9.  Null Hypothesis: The pesticide will not significantly reduce the survivorship of Escherichia coli or Staphylococcus epidermidis.  Alternative Hypothesis: The pesticide will significantly reduce the survivorship of Escherichia coli or Staphylococcus epidermidis.

10.  Sterile test tubes  Micropipettes  Bayer Pesticide (0.015% β-cyfluthrin, 0.012 % imidacloprid)  Vortex  Ethanol  Spreader  LB Agar Plates  LB Media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)  Incubator  Labeling Tape  Marker  Escherichia coli (DH5- Alpha)  Staphylococcus epidermidis  Test tube rack  Sidearm flask  Sterile filters  Klett Spectrophotometer  Pipette tips  Sterile Dilution Fluid (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl)  Bunsen Burners  Matches  Latex gloves

11. 1. Escherichia coli and Staphylococcus epidermidis were grown overnight in sterile LB media. 2. A sample of those cultures were added to media in a sidearm flask. 3. The cultures were incubated until a density of 50 Klett spectrophotometer units was reached. This represents a density of 10 8 -10 9 cells/ml. 4. This cultures were diluted in sterile dilution fluid to a density of 10 5 cells/ml. 5. The pesticide was sterile filtered and was diluted to concentrations of 0%, 0.01%, 0.1%, and 1% in sterile dilution fluid.

12. 0% Pesticide 0.01% Pesticide 0.1% Pesticide 1% Pesticide SDF9.9 mL9.899 mL9.89 mL9.8 mL Pesticide0 mL0.001 mL0.01 mL0.1 mL E. Coli/Staph 0.1 mL Total10 mL

13. 6. 0.1 mL of the E. coli culture was added to 4 tubes of each concentration of pesticide and 0.1 mL of the Staphylococcus was added to 4 tubes of each concentration of pesticide. This produced a final volume of 10 mL, and a cell density of 10 3 cells/ml. 7. After vortexing to evenly suspend cells, 0.1 mL aliquots from each tube was spread evenly on LB agar plates. 8. The plates were incubated at 37 °C overnight. 9. The resulting colonies were counted. Each colony is assumed to have risen from one cell.

14. 1. Sterilized pesticide was spread evenly over LB agar plates in two concentrations, a stock concentration (200 µL of pesticide) and a 10% concentration (20 µL of pesticide and 180 µL of sterile dilution fluid.) 2. The plates were incubated at 37 °C for two hours. 3. Escherichia coli and Staphylococcus epidermidis were grown overnight in sterile LB media. 4. 100uL aliquots of Control cell suspension were spread onto the infused plates. 5. The plates were incubated at 37 °C overnight. Resulting colonies counted.

15. P value: 8.19E-06

16. P value: 2.23E-06

17. Pesticide concentration T valueInterpretation 0.01%2.47Not significant 0.1%4.43Significant 1%9.06Significant T critical = 3.5 Alpha =.05 Pesticide concentration T valueInterpretation 0.01%4.03Significant 0.1%7.98Significant 1%9.72Significant Escherichia coli Staphylococcus epidermidis

19. P-value:1.8E-09 P-value:1.07E-08

20. Pesticide concentration T valueInterpretation Low4.16Significant High10.93Significant T critical = 2.67 Alpha =.05 Pesticide concentration T valueInterpretation Low5.3Significant High9.97Significant Escherichia coli Staphylococcus epidermidis

21. A. Liquid Exposure The null hypothesis that pesticide will not affect E. coli or Staphylococcus epidermidis survivorship was rejected for all of the concentrations of pesticide, except for the 0.01% exposure to E. coli. Evidence suggests that the pesticide significantly reduces the survivorship in both species. B. Agar Infusion The null hypothesis was rejected. Evidence suggests that the pesticide significantly reduces the survivorship in both species.

22. Limitations  It was difficult to exactly synchronize the plating for the microbes, possibly contributing to the varying colony counts.  Infusion of pesticide into LB agar was not exact. Extensions and Future Studies  Higher and lower concentrations of pesticide  Different types of pesticides  More species of bacteria  More replicates  Liquid agar infusion  Vary pesticide exposure time  Perform a growth rate experiment

23.  http://www.epa.gov/opp00001/pestsales/07 pestsales/market_estimates2007.pdf  http://www.epa.gov/pesticides/about/  http://www.green- networld.com/facts/pollution.htm  http://www.beyondpesticides.org/pesticides/ factsheets/Synthetic%20Pyrethroids.pdf  http://www.docstoc.com/docs/9271628/Effe ct-of-pesticides-on-environment

24. SUMMARY GroupsCountSumAverageVariance 0%41238309.5127 0.01%41161290.2580.25 0.1%41100275124 1%4956239153.3333 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups10686.1933562.06329.40318.19E-063.490295 Within Groups1453.7512121.1458 Total12139.9415

25. SUMMARY GroupsCountSumAverageVariance 0%4913228.25188.25 0.01%4764191270.6667 0.1%4618154.5203.6667 1%4554138.519.66667 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups19226.1936408.72937.574082.23E-063.490295 Within Groups2046.7512170.5625 Total21272.9415

26. SUMMARY GroupsCountSumAverageVariance Control81587198.375114.2679 Low81428178.557.14286 High81169146.125102.4107 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups11128.5825564.29260.962631.8E-093.4668 Within Groups1916.752191.27381 Total13045.3323

27. GroupsCountSumAverageVariance Control82671333.875166.4107 Low82309288.625304.8393 High81990248.75403.3571 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups29023.58214511.7949.777061.07E-083.4668 Within Groups6122.2521291.5357 Total35145.8323