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

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

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

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

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

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.

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.

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

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.

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.

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

LD 50- 197.5

LD 50- 103.5

Staph Dunnett's Test T-crit=3.48 25% 50% 75% 99% T-value -10.721 -22.885 -30.913 -34.375 Accept/ Reject Null Reject Null

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

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

Limitations Time Resources Synchronizing of the plates

Extensions More replicates More concentrations

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

References http://www.cdc.gov/bloodsafety/bbp/diseases_organisms.html http://www.cdc.gov/ecoli/ http://www.webmd.com/a-to-z-guides/e-coli-infection-topic-overview

Staph ANOVA

E. coli ANOVA