1. Microbial Survivorship in River Water
Timothy McClelland
Grade 9
Pittsburgh Central Catholic High School

2. The Three Rivers Second most diverse river system
Used to be of industrial pollution and disease
Bad for the public health and safety

3. The Clean Water Act
Passed due to the growing public awareness and concern for controlling water pollution
Gave EPA the authority to implement pollution control programs
Sets water quality standards for all contaminants in surface waters

4. Staphylococcus epidermidis
Bacteria found on healthy human skin
Forms 1/2-2 mm. bio films
Commonly used model for microbial flora

5. Escherichia coli One of the most common forms of bacteria
Found in intestinal tracts of many mammals
Most studied prokaryote in biological research
Are many of different strains; most non-pathogenic

6. Purpose
Did the Clean Water Act help to clean river water in Pittsburgh?
To asess the survivorship of E. coli and Staphylococcus epidermidis, in the Pittsburgh region, specifically in the Allegheny river.

7. Hypothesis
Alternative- River water concentrations will not significantly decrease survivorship of E. coli and Staphylococcus epidermidis.
Null- River water concentrations will significantly decrease survivorship of E. Coli and Staphylococcus epidermidis.

8. Materials
LB agar plates(1% tryptone, 0.5% yeast extract, 1% NaCl, 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) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)
E.coli
Staphylococcus epidermidis (Staph)
0.22 micron syringe filters + 10 mL syringe
Allegheny river water

9. Procedure
Staphylococcus Epidermidis and E. coli were grown overnight in sterile LB media.
Samples of the overnight cultures were added to fresh media in a sterile sidearm flask.
The cultures were diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
Varying mL of sterile river water (0.22 micron syringe filtered) and varying mL of sterile water were transferred to sterile 20 mL capped test tubes.
100 µL of cell culture was then added to the test tubes, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.

10. Procedure (Cont'd)
The solutions were mixed by vortexing and allowed to sit at room temperature for 15 minutes.
After vortexing to evenly suspend cells, 100 µL aliquots were removed from the tubes and spread on LB plates.
The plates were incubated for 48 hours.
The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

11. Concentrations Control 25% 50% 75% 99% 0 mL 2.5 mL 5 mL 7.5 mL 9.9 mL
Variable
7.4 mL
4.9 mL
2.4 mL
Sterile Water
.1 mL
E. Coli & Staph
10 mL
Total mL

12. LD

13. LD

14. Staph Dunnett's Test T-crit=3.48 25% 50% 75% 99% T-value -10.721
Accept/ Reject Null
Reject Null

15. E. coli Dunnett's Test T-crit=3.48 25% 50% 75% 99% T-value 2.498
10.751
4.428
1.482
Accept/ Reject Null
Accept Null
Reject Null

16. Conclusions Reject the null hypothesis
Increased the survivability of E. coli
Decreased the survivability of Staph but was not significant

17. Limitations
Time
Resources
Synchronizing of the plates

18. Extensions
More replicates
More concentrations

19. Future Research River water affect on other bacteria and microbes
River water from different parts of the river
Survivorship of E. coli and Staph in water of other rivers

20. References http://www.cdc.gov/bloodsafety/bbp/diseases_organisms.html

21. Staph ANOVA

22. E. coli ANOVA