Microbial Survivorship in River Water John Crelli Grade 10 Pittsburgh Central Catholic High School

The Clean Water Act Growing public awareness and concern for controlling water pollution led the government to pass the Clean Water Act. It gave EPA the authority to implement pollution control programs such as setting wastewater standards for industry. Also continued requirements to set water quality standards for all contaminants in surface waters. It is unlawful for any person to discharge any pollutant from a point source into navigable waters, unless a permit was obtained.

The Three Rivers Allegheny, Monongahela, and Ohio A unique confluence of river systems unlike any other in the world, diverse and more abundant than any other state except Alaska. For more than a century, Pittsburgh's three rivers were an unsightly cesspool of industrial pollution and disease and avoiding them at all costs was a matter of public health and safety. Finally, thanks to the Clean Water Act, mentioned earlier, Pittsburgh’s rivers are clean again.

Purpose Did the Clean Water Act help to clean river water in Pittsburgh? Even if the water is clean, can bacteria still survive? Can eukaryotic organisms survive even if prokaryotic organisms can’t? The purpose of this experiment is to assess the survivorship duration of a bacterial model, E. coli, and an eukaryotic model, Saccharomyces cerevisiae, in the Pittsburgh region, specifically in the Monongahela and Allegheny rivers.

E. Coli Escherichia coli (E.coli) is one of the most common forms of bacteria found in many environments including the intestinal tracts of many mammals. E.coli has also been utilized as the most studied prokaryote in biological research. There are many of different strains of E.coli, most of which are non- pathogenic. However, there are strains which can produce fatal disease in humans and other mammals.

Saccharomyces cerevisiae The yeast species Saccharomyces cerevisiae has been used in baking and fermenting alcoholic beverages for thousands of years. It is also important as a model organism in modern cell biology research, as it is the most thoroughly researched eukaryotic microorganism. Scientists have used Saccharomyces cerevisiae for information on the biology of the eukaryotic cell.

Past Studies 1 E. coli survivorship was tested in spring water in Arkansas. In a period of 75+ days, there was a 4-log (10 4 ) die-off.

Past Studies 2 Yeast survivorship was observed in the Tagus estuary in Portugal. Yeast was still able to grow even with tides and river discharge.

Null and Alternative Hypotheses River waters will not significantly affect the survivorship of E. coli or Saccharomyces cerevisiae. The survivorship of E. coli and Saccharomyces cerevisiae populations will be altered in river water.

Materials 54 LB agar plates(1% tryptone, 5% yeast extract, 1% NaCl, 2 mL 1M NaOH, 1.5% agar) 54 YEPD agar plates (1% yeast extract, 2% peptone, 2% glucose (dextrose), 1.5% agar) YEPD Media (1% yeast extract, 2% peptone, 2% glucose (dextrose)) LB media (1% tryptone, 5% yeast extract, 1% NaCl) Klett spectrophotometer 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 KH 2 PO 4, 10 mM K 2 HPO 4, 1 mM MgSO 4, 0.1 mM CaCl 2, 100 mM NaCl) E.coli B Saccharomyces cerevisiae (Yeast) 0.22 micron syringe filters + 10 mL syringe 2 liters Monongahela River and Allegheny River water 2 liters Allegheny River water

Procedure 1. Saccharomyces cerevisiae and E. coli were grown overnight in sterile YEPD and LB media. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in incubators (30°C and 37°C) until a density of 50 Klett spectrophotometer units were reached. This represents cell densities of approximately 10 7 cells/mL and 10 8 cells/mL. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL mL of sterile river water(0.22 micron syringe filtered) were transferred to sterile 20 mL capped test tubes. 9.9 mL of SDF were added to the control test tubes µL of cell culture was then added to the test tubes, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7. The solutions were mixed by vortexing and allowed to sit at room temperature for 15 minutes. 8. After vortexing to evenly suspend cells, 100 µL aliquots were removed from the tubes and spread on YEPD and LB plates. 9. The plates were incubated at 30 and 37 degrees for 48 and 24 hours. 10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

Statistical Analysis P-valuesTime PeriodAlleghenyP-valuesMonongahela P = min P <.01P = P >.05 P = P = P = 5.48E min P <.01P = P <.05 P = P <.01P = P >.05 P = min P <.01P = P <.01 P = 2.65E-08P <.01P = P <.01

Conclusions At 0-10 minute exposure, the Allegheny river water had a statistically significant effect on Yeast survivorship (+). The Monongahela river water did not. At 30 minute exposure, the Allegheny river water had a statistically significant effect on Yeast (+) and E. coli (-) survivorship. The Monongahela river water had a statistically significant effect on Yeast survivorship (-), but not on E. coli survivorship. At 60 minute exposure, the Allegheny and Monongahela river water both had a statistically significant effect on Yeast (+) and E. coli survivorship.

Limitations/Extensions There is a lag between pipetting and plating, so that the exact times of exposure to the variable are not quite consistent. This could be remedied by having a team of students perform these procedures simultaneously or by carefully timing the plating technique. River waters will be infused into the agar plates. Although 6 trials were employed for each of the rivers and the control, more trials are suggested. This laboratory model will be extended to include the Ohio River. A longer duration of data collection might also reveal more long term or subtle effects.

Sources Blackwell Synergy (Blackwell-synergy.com) EPA (Epa.gov) Dr. John Wilson, University of Pittsburgh Biostatistician Wikipedia (Wikipedia.com)