Microbial Survivorship in River Water

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

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. Past Studies 1 • E. coli survivorship was tested in spring water in Arkansas. • In a period of 75+ days, there was a 4-log (104) die-off.

8. 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.

9. 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.

10. 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 KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 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

11. Procedure • Saccharomyces cerevisiaeand E. coli were grown overnight in sterile YEPD and LB media. • Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. • 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 107 cells/mL and 108 cells/mL. • The cultures were diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL. • 9.9 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. • 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. • 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 YEPD and LB plates. • The plates were incubated at 30 and 37 degrees for 48 and 24 hours. • The resulting colonies were counted. Each colony is assumed to have arisen from one cell.