1. Patrick Doyle Pittsburgh Central Catholic Grade 9

2.  Runoff is a part of the water cycle and describes the water that flows over a land surface.  Surface runoff that occurs on land typically creates a “watershed.” ◦ Typical materials transported: fertilizers, petroleum, pesticides, herbicides, and salts. ◦ Runoff often leads to a disruption of aquatic ecosystems.

3.  “Table Salt”  Used on roads during winter to prevent ice formation  Chemical formula NaCl

4.  Commonly found in fertilizers  Chemical formula KCl  Sodium free substitute to common table salt  Used in some chemical deicers

5.  PennDOT used 831,000 tons of salt in the past 5 years ◦ This figure represents only main roads and bridges that were salted by PennDOT, not other roads salted by private companies Question: How does salt runoff impact microbial populations in fresh water aquatic systems? Are bacterial populations disrupted?

6.  Escherichia coli is a large and diverse group of gram (-) bacteria.  It is found in the intestinal tract of most animals, including humans  Most strains of E. coli are harmless, others cause sickness.  Estimated to cause infection in more than 70,000 patients a year in the United States.  Common in aquatic systems  Serves as a common prokaryotic cell model.

7.  Bacteria usually non-pathogenic towards humans  Commonly found on the epidermis of humans  Common prokaryote model  Gram + bacteria

8. Gram PositiveGram Negative Cytoplasmic lipid membrane Cytoplasmic membrane Thick Peptidoglycan layer Thin Peptidoglycan layer S. epidermidis is the gram positive model E. coli is the model for Gram negative

9.  Show that salt runoff caused severe dehydration in roadside plant life  This occasionally resulted in the death of plant

10.  To determine the effects of Sodium and Potassium Chloride on gram positive and negative bacteria.

11.  Null Hypothesis: Salt will not have an effect on the survivorship of either bacterial species  Alternative Hypothesis: Salt will reduce the survivorship of both bacterial species

12.  LB agar plates (1% trytone, 5% yeast extract, 1% NaCl, 2mL 1M NaOH, 1.5% agar)  LB media (1% tryptone, 5% yeast extract, 1% NaCl)  Sterile pipette tips  Micropipettes  Vortex  Incubator  Sidearm flask  Spreading platform, spreader bar, ethanol  20 mL Sterile capped test tubes with Sterile Dilution Fluid (SDF) (10mM KH 2 PO 4, 10 mM K 2 HPO 4, 1mM MgSO 4, 0.1 mM CaCl 2, 100 mM NaCl)  Escherichia coli  Staphylococcus epidermidis  25% stock solutions of NaCl and KCl  0.22 micron syringe filters 10ml syringe

13. 1. Bacteria 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 cultures were grown to a density of 50 Klett spectrophotometer units (approximately 10^8 cells/ml 4. Cultures were diluted in SDF to approximately 10^5 cells/ml 5. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL. 6. The tubes were prepared as follows :

14. 0%0.1%1%10% Sterile Dilution Fluid 9.9ml9.86ml9.5ml5.9ml Microbe.1ml Salt Solution0ml.04ml.4ml4ml Chart of Concentrations

15. 7. 100 µL of cell culture was then added to the salt 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

16. P- Value=2.99E-11 Alpha=.05

17. P Value= 9.35E-17

18. Alpha=.05 P- Value=2.05E-12

19. Alpha=.05 P- Value=2.82E-11

20. S. Epidermidis E. Coli Salt Concentration T-Value Interpretation Salt Concentration T-Value Interpretation 0.1% NaCl 4.24 Significant 0.1% NaCl3.01 Insignificant 1% NaCL13.9 Significant 1% NaCl7.9Significant 10% NaCl14.69 Significant 10% NaCl14.37 Significant 0.1% KCl2.86 Insignificant 0.1% KCl9.49 Significant 1% KCl7.77 Significant 1% KCl21.1 Significant 10% KCl14.14 Significant 10% KCl27.44 Significant T-Crit 3.10 Dunnett’s Test

22. 1. The salt solutions were infused by spreading 200 µl of the salt stock solution onto the agar surface. The plates were then inverted and placed in an incubator for 2 hours for infusion 2. E. coli was grown overnight in sterile LB media. 3. E. coli suspensions were transferred from the control tube in experiment 1to the salt infused plates 4. The plates were incubated at 37 C for 24 hours. 5. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

23. SIG

24.  Null Hypothesis can be Rejected ◦ However it cannot be rejected for the.1% concentrations of E. coli NaCl and S. Epidermidis KCl.  Alternative Hypothesis is supported. ◦ The majority of concentrations significantly reduced the survivorship of the bacteria. ◦ The LD-50 showed that NaCl was more effective at killing S. Epidermidis.

25.  Limitations ◦ During plating exposure to the salt varied slightly. ◦ Limited number of concentrations of salt tested. ◦ Synchronization of plating difficult to achieve. ◦ Only one exposure time was used.  Extensions and Future Studies ◦ Different strains of bacteria can be used ◦ The same salts effect on plant life can be incorporated ◦ Wider salt concentration range. ◦ Test synergistic effects of salt with other factors.

26.  http://www.drugs.com/pro/potassium- chloride.html  http://hyperphysics.phy- astr.gsu.edu/hbase/molecule/nacl.html  http://www.greenviewfertilizer.com/articles/un derstanding-N-P-K  http://www.medicalnewstoday.com/articles/685 11.php  http://web.uconn.edu/mcbstaff/graf/Student%2 0presentations/S%20epidermidis/sepidermidis.h tml  http://www.life.umd.edu/classroom/bsci424/La bMaterialsMethods/GramStain.htm  http://onlinepubs.trb.org/onlinepubs/sr/sr235/ 069-082.pdf

27. Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Row 1614223.66667127.4667 Row 2650984.8333352.56667 Row 365709580.4 Row 4657195.1666798.96667 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups21228.3337076.11178.754712.99E-113.098391 Within Groups17972089.85 Total23025.3323