1. Effects of Road Salt Deicers on Yeast Survivorship James Brunner Pittsburgh Central Catholic High School Grade 9

2. Introduction  In this experiment, a yeast culture was tested under the effects of various chloride salts  Model- yeast, Variable- chloride salts, Interaction- yeast survivorship  Idea arose from wondering what effects salting roads has on local ecosystems

3. Road Salt Runoff  In PA, PENNDOT uses 96,367 tons of salt on roads in winter  Several salts used as deicers  These salts have harmful effects on everything from plants and animals to concrete and metals

4. Sodium Chloride  Standard salt used to de-ice roads  Known damages- harmful to water, plants, metals, and even the roads it’s used on  One of the cheapest salts to melt ice  Chemical Symbol: NaCl

5. Magnesium Chloride  Also used as road salt de-icer  Less harmful then Sodium Chloride  Known to damage metal and concrete, but still known as best for metal and concrete  Contains 17-56% more chloride ion than other salts  Melts ice at slower rate than other chloride salts  Chemical Symbol: MgCl 2

6. Potassium Chloride  Another salt used as de-icer  Thought to be one of the “safer” salts to melt ice with  Used as sodium-free substance for table salt  Known as best for use with vegetation  Chemical Symbol: KCl

7. Yeast  Eukaryotic organism used in foods and drinks  Most commonly studied organism in labs  Easy to use- grows quickly, easy to count colonies, and nonpathogenic (safe to use)  Less tolerable to extreme conditions- achieves better data

8. Past Studies  Several studies of the nature of this experiment have been conducted in the past, such as: Effects of NaCl on yeast Effects of NaCl on macro-invertebrates  In both studies, it was found that NaCl had a negative effect on the organisms’ survivorship

9. Purpose & Hypotheses  Purpose- to determine the effects of various chloride salts on a yeast culture  Null Hypothesis- The survivorship of the yeast cells will not be significantly affected by any of the concentrations of the three chloride salts  Alternative Hypothesis- all three chloride salts will reduce survivorship compared to the control

10. 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))  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)  Saccharomyces cerevisiae (Yeast)  0.22 micron syringe filters + 10 mL syringe  Weigh boat  Scale  Incubator  Vortex Genie  Stir Plate  NaCl (10%)  KCl (10%)  MgCl 2 (10%)  Pipettes  Sterile test tubes  Water  Ethanol (95%)  Spreader Bar  Micro Burner

11. Procedure 1. Yeast was grown overnight in sterile YEPD media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in an incubator(30 Degrees) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/ml. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/ml. 5. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations for a total of 9.9 ml. For Example:1 ml of 10% variable ‘z’ solution + 8.9 ml of SDF= final concentration of almost 1% ‘z’ (the addition of 0.1 ml of cell culture will result in a total of 10 ml and a 1% concentration). 6. 10 3 100 ml 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 solution was mixed by vortexing and allowed to sit at room temperature. 8. After vortexing to evenly suspend cells, 0/1 ml aliquots were removed from the tubes and spread on YEPD+LB plates. 9. The plates were incubated at 30 degrees for 48 hours. 10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

12. Test Tube Contents 0%0.1%0.5%1% SDF (ml) 9.99.89.48.9 10% Salt Stock (ml) 00.10.51 Yeast (ml) 0.1 Total Volume (ml) 10

13. Results  All salts affected the yeast; yeast survivorship declined as salt concentration rose  NaCl was the least harmful, while MgCl 2 was the most harmful  All plates grew yeast well; except for the plates containing.5% KCl, which was contaminated and did not grow any yeast  The results in this experiment pertained to those of past studies of this nature

14. Effects of Salt on Yeast Survivorship #ofcolonies#ofcolonies Type of Salt Salt Concentration 0.02 0.04 0.03 = P values lower then 0.05

15. Pictures NaCl 1%KCl.1% MgCl 2.5%No Salt Added

16. ANOVA Chart

17. Conclusions  After the data was collected, the following concentrations of salt rejected the null hypothesis: 1%KCl, and 1% MgCl 2  All colonies affected negatively by the salts  Alternative Hypothesis correct- all salts reduced yeast survivorship  According to data, road salt de-icers inhibit the growth of yeast colonies

18. Conclusions (continued)  After ANOVA and Dunnett tests, null hypothesis rejected  Most ANOVA tests showed null hypothesis accepted, with no significant difference from control (most p values over.05), but Dunnett test showed that it was rejected, with significant difference from control (most t values less than t critical)  Dunnett test more powerful than ANOVA- compares better over two groups of data  ANOVA tests may have shown that null was accepted as decreases in yeast survival not very large, but clearly exist

19. Limitations and Extensions  All.5% KCl contaminated-data could not be obtained  Difficult to ensure exact exposure times to salt variables due to time variation in spread plating. This could be more easily overcome by using a team of people  More concentrations and various salts could be tested  Other cellular models could be tested  Another model, such as bacteria, added to achieve better data

20. References  “Rock Salt (NaCl) Sodium Chloride.” Peterschemical.com Dec. 21, 2007. <http://www.peterschemical.com/sodium-chloride/  “Potassium Chloride and Urea.” Peterschemical.com Dec. 21, 2007. < http://www.peterschemical.com/potassium-chloride/  “Magnesium Chloride.” Peterschemical.com Dec 21, 2007. <http://www.peterschemical.com/magnesium-chloride/  “Yeast.” Wikipedia.org Dec. 21, 2007. <http://en.wikipedia.org/wiki/Yeast  “Sodium Chloride.” Wikipedia.org Dec. 21, 2007. <http://en.wikipedia.org/wiki/Sodium_chloride  “Magnesium Chloride.” Wikipedia.org Dec. 21, 2007 <http://en.wikipedia.org/wiki/Magnesium_chloride

21. References (continued)  “Potassium Chloride.” Wikipedia.org Dec. 21, 2007 <http://en.wikipedia.org/wiki/Potassium_chloride  “Road Salt and Water Quality.” Des.state.nh.us Dec. 21, 2007 <http://www.des.state.nh.us/factsheets/wmb/wmb-4.htm  “Field and laboratory investigations on the effects of road salt (NaCl) on stream macroinvertebrate communities.” Sciencedirect.com Dec. 21, 2007 <http://www.sciencedirect.com/science?_ob=ArticleURL&_u di=B6VB5- 455VM763&_user=10&_rdoc=1&_fmt=&_orig=search&_sort =d&view=c&_acct=C000050221&_version=1&_urlVersion= 0&_userid=10&md5=c9d9b919f172d211bbad1839fd5d0afe  “Effect of Sodium Chloride on Bakers' Yeast Growing in Gelatin.” Aem.asm.org Dec. 21, 2007 <http://aem.asm.org/cgi/content/abstract/43/4/757  “Winter Maintenance- Notes and Other Interesting Information” Dot.state.pa.us Jan. 28, 2008. <http://www.dot.state.pa.us/Penndot/Districts/district6.nsf/winter_ maint_notes?ReadForm