1. Rainwater Toxicity Effects on Microbial Life. Phillip Ferguson February 2, 2008 Central Catholic High School

2. Water Pollution Contributed to by neighborhood people (fertilizers, unclean gutters as a source of debris, other trash items and residue to water sources). Affects water dwelling organisms (polluting of the water leads to harming the fish population, which leads to harming of sea-faring birds. Surface runoff pollutes lakes, streams, and rivers (Silt from logging operations, farmland feces, chemical waste from large industries).

3. Types of Pollutants Organic Pollutants – Bacteria found in animal feces, tree and brush debris from logging camps, processed food from landfills. Inorganic Pollutants – Silt runoff from construction sites, chemical waste from industrial products, heavy metals from acid mine drainage.

4. Water Cycle

5. Affects of Farmland Feces As rainwater runs through farmland, it may pick up E coli bacteria. The more E coli the rainwater absorbs, the more that gets into water supplies. As the amount of E coli in water increases, the more of a chance there is that the bacteria gets into tap water and in turn infects people.

6. First Flush System designed to collect the first wave of rainwater from rooftop gutters. Intercepts harmful debris and stops it from getting to water sources and polluting water supplies.

7. Yeast Growth form of eukaryotic microorganisms classified in the kingdom Fungi. Has been widely used in genetics and cell biology, largely because the cell cycle in a yeast cell is very similar to the cell cycle in humans.

8. Purpose and Hypothesis To investigate the effects of local rainwater on the survivorship of yeast colonies. This could help gauge the toxicity or other inhibitory effects of rainwater. Hypothesis – The survivorship of yeast colonies will be significantly affected by all the tested concentrations of rainwater. Null Hypothesis – The survivorship of yeast colonies will not be significantly affected by the tested concentrations of rainwater.

9. Materials YEPD agar plates (1% yeast extract, 2% peptone, 2% glucose (dextrose), 1.5% agar) YEPD Media (1% yeast extract, 2% peptone, 2% glucose (dextrose) Klett spectrophotometer Sterile pipette tips Micropipettes Vortex Incubator Sidearm flask Spreading platform, spreader bar, ethanol Sample rainwater (stock) 0.22 micron syringe filters + 10 mL syringe Distilled water  Ethanol  Bunsen burner  Incubator Saccharomyces cerevisiae (Yeast)  Timer or clock  700 mL rainwater sample from Fox Chapel PA

10. Procedure  S.c. Yeast was grown overnight in sterile YEPD Media.  A sample of the overnight culture was added to fresh media in a sterile sidearm flask.  The culture was placed in a shaking water bath (30 0 C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/ml.  The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/ml. The rainwater samples were sterile filtered using 0.22 micron syringe filters. Concentrations of stock (rainwater), yeast, and distilled water were prepared in 10 test tubes using 5 different varieties of concentrations (two test tubes per concentration were arranged in rows with the corresponding concentration vertically aligned with its twin). After concentrations are prepared, wait 15 minutes.

11. Procedure (con.) After 15 mins,0.1 mL aliquots were spread onto YEPD auger plates from each concentration in the first row of tubes to 8 auger plates each. After 45 mins, 0.1 mL aliquots were spread onto YEPD auger plates from each concentration in the second row of tubes to 8 auger plates each.  The plates were incubated at 30 0 C for 48 hours.  The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

12. Concentrations for Procedure Tubes1, 23, 45,67,89,10 Stock (mL) 9.9 [~100 % ] 5 [50%] 2.5 [25%] 1 [10%] 0 [0%] Distilled Water (mL) 04.97.48.99.9 Yeast (mL) 0.1 Total (mL) 10

13. p = 1.55202E-13 P <.05

14. P = 9.24553E-10 P <.05 P >.05 P <.05

15. Conclusion After conducting both an Anova and Dunnett’s Test for the data, it was concluded that the 15 minute set of data was significantly affected by the stock (rainwater), and the null hypothesis was rejected. Surprisingly, the yeast colonies were all positively affected by the concentrations of stock.

16. Conclusion (con.) After 45 minutes of exposure it was discovered that both the 100% and 10% concentrations significantly affected the yeast colony population. The 100% stock negatively influenced the yeast colonies, while the 10% stock positively influenced yeast colony population.

17. Limitations and Flaws Just one test subject (only yeast) as opposed to more (yeast and bacteria). Difficult to get the exact same time of exposure for each sample because of lag time between transfer of stock between auger plates and test tubes.

18. Extensions Use two kinds of test subjects (yeast and bacteria). Take sample rainwater from different areas. Expose the test subjects to the stock for different amounts of time (elongate exposure to ensure encompassing results). Fuse the rainwater into the auger plates to more evenly distribute the rainwater.

19. References http://en.wikipedia.org/wiki/Rainwater http://en.wikipedia.org/wiki/Yeast http://en.wikipedia.org/wiki/Water_pollution http://www.waterencyclopedia.com/En- Ge/Fresh-Water-Nautral-Composition-of.html.