1. Patrick Marchione Central Catholic High School Grade 9

2.  First sports drink created by a chemist, William Owen, in the U.K. in 1927.  In 1968, sports drinks began to revolutionize sports.  Used for rapid replacement of electrolytes and fluid lost during exercise.  Restores depleted muscle carbohydrate storages that cause muscle failure and fatigue. Sports Drinks

3.  Water  Glucose  Sucrose  Dextrose  Total Carbohydrates 34g  Citric acid  Natural flavor  Sodium citrate  Monopotassium phosphate  Gum arabic  Red 40  Glycerol ester of rosin Gatorade Ingredients  Protein 0g  Sodium 270 mg  Potassium 75mg  Total Fat 0g

4. Vitamin Water Ingredients  Reverse osmosis water  Crystalline fructose  Cane sugar  Taurine 25 mg  Total Carbohydrate 32g  Total Fat 0g  Sodium 0g  Protein 0g  Less than 0.5% of:  Vitamin C  Vitamin B12  Natural flavors  Vegetable juice  Magnesium lactate  Chromium polynicotinate  Citric acid  Vitamin B6  Calcium lactate  Potassium phosphate  Vitamin B5  Zinc gluconate

5. Micro Bacterial Flora  Present in the respiratory, urinary, and digestive tracts as well as on skin.  Consists of mostly bacteria, few protists and fungi.  Most are harmless, but some can cause diseases.  Influences human anatomy, physiology, and overall health.  10 14 bacteria are harbored within and on humans.  Many products are tested for effects on human cells. Micro Flora Effects?

6.  Gram positive coccus  Common surface symbiont in many mammals (including humans)  Most forms considered non-pathogenic.  Potentially pathogenic upon systemic entry  Forms biofilms Staphylococcus epidermidis

7.  Rod-shaped, 2 micrometers diameter  Gram negative bacteria  Reproduction rate: 20 min  Survival, growth, and replication require only a single carbon source and ammonium salts.  Common symbiont found in the lower intestines of warm blooded animals.  Few pathogenic - Almost 73,000 cases of infection and 61 deaths per year in the United States.  Common prokaryote model Escherichia coli

8. To determine the effects of Gatorade and Vitamin Water on S. epidermidis and E. coli survivorship. Purpose

9. Null Hypothesis Gatorade and Vitamin Water will not have a significant effect on the survivorship of E. coli and S. epidermidis Alternate Hypothesis Gatorade and Vitamin Water will significantly alter the survivorship of E. coli and S. epidermidis

10. Materials Gatorade sports drink Vitamin Water sports drink Mirco pipettes + Sterile tips Spreader bars 60 LB agar plates(1% Tryptone, 0.5% Yeast extract, 1% NaCl) Escherichia coli Dh5- alpha (E. Coli) (Obtained from Doonan Lab, CMU) Staphylococcus espidermidis (Staph) (Obtained from Ward’s Scientific) Burner Sidearm Flask Vortex Klett Spectrophotometer SDF (Sterile Dilution Fluid) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) 8 sterile test tubes Ethanol

11. 1. Bacteria (E. coli and Staph) were grown overnight in sterile LB media. 2. Samples of the overnight culture were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in incubators at 37°C until a density of 50 Klett spectrophotometer units were reached. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10E5 cells/mL. 5. Various concentrations of two different sports drinks (0%, 1%, 10%, and 50%) were created in test tubes containing SDF, resulting in 10 mL per tube Procedure (Liquid Exposure)

12. Concentration Chart E. coli/Staph0.1 mL Variable0 mL0.1 mL1 mL5 mL SDF9.9 mL9.8 mL8.9 mL4.9 mL Concentration[0%][1%][10%][50%]

13. 6. 100 µL of cell culture was then added to the test tubes, yielding a final volume of 10 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 were removed from the tubes and spread on LB plates. 9. The plates were incubated at 37°C for 24 hours. 10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell. Procedure (Liquid Exposure continued)

14. Anova Statistical test that allows the comparison of different groups (between group variation vs. within group variation) Dunnett’s test A follow up statisical test for the comparison of a particular variable group vs. the control group to determine significant variation Anova and Dunnett’s Test

15. Sport Drink Effects on E. coli Survivorship P value 0.000134 P value 0.00017

16. Sport drink effects on S. epidermidis Survivorship P value 0.068637 P value 1.7E-05

17. T-crit= 3.29 Dunnett’s Test E. coli Gatorade Concentration T valueSignificance [1%] 0.97Not Significant [10%] 2.24Not Significant [50%] 6.42Significant

18. T-crit= 3.29 Dunnett’s Test E. coli Continued.. Vitamin Water Concentration T valueSignificance [1%] 1.33Not Significant [10%] 2.98Not Significant [50%] 6.71Significant

19. T-crit= 3.29 Dunnett’s Test S. epidermidis Gatorade Concentration T valueSignificance [1%] 4.36Significant [10%] 6.93Significant [50%] 8.06Significant

20.  The null hypothesis can be accepted for:  All concentrations of Vitamin Water [E. coli]  1%, 10% of Gatorade [E. coli]  1%, 10% of Vitamin Water [Staph]  The null hypothesis can be rejected for:  All concentrations of Gatorade [Staph]  50% Vitamin Water [Staph]  50% Gatorade [E. coli] Conclusions

21. Limitations ❖ Plating slightly unsynchronized ❖ Only 4 concentrations of variables tested ❖ Only one kind of test, Liquid Pulse Extensions ❖ Use more types of sports drinks ❖ Use more concentrations ❖ Agar Infusion ❖ Synergistic effects

22.  "Escherichia Coli." MicrobeWiki. Kenyon.edu, n.d. Web. 20 Dec. 2012.  Orenstein, Abigail. "The Discovery and Naming of Staphylococcus Aureus." Antimicrobe.org. N.p., n.d. Web. 20 Dec. 2012.  Todar, Kenneth, PhD. "Staphylococcus Aureus." Staphylococcus Aureus. N.p., 2008. Web. 20 Dec. 2012.  "Sports Drinks: The Real Facts." Healthportal. N.p., n.d. Web. 28 Jan. 2015.  "Who Invented Sports Drinks? - HowStuffWorks." HowStuffWorks. N.p., n.d. Web. 28 Jan. 2015. References

23. Anova- alpha 0.05 Anova: Single FactorVitamin Water E. coli SUMMARY GroupsCountSumAverageVariance Column 142439609.75710.9167 Column 242294573.5137 Column 342114528.5217 Column 4417084274876.667 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups75102.69325034.2316.853570.0001343.490295 Within Groups17824.75121485.396 Total92927.4415

24. Anova- alpha 0.05 Anova: Single FactorGatorade E. coli SUMMARY GroupsCountSumAverageVariance Column 1426686671162 Column 242548637556 Column 342391597.751154.917 Column 4418724684805.333 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups92233.19330744.416.016360.000173.490295 Within Groups23034.75121919.563 Total115267.915

25. Anova- alpha 0.05 Anova: Single FactorVitamin Water Staph SUMMARY GroupsCountSumAverageVariance Column 143649186.66667 Column 2436390.7557.58333 Column 3432280.586.33333 Column 4430676.549 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups644.68753214.89583.0745160.0686373.490295 Within Groups838.751269.89583 Total1483.43815

26. Anova- alpha 0.05 Anova: Single FactorGatorade Staph SUMMARY GroupsCountSumAverageVariance Column 14507126.75128.9167 Column 2438395.7573.58333 Column 3431077.567 Column 4427869.5135 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups7750.2532583.41725.546771.7E-053.490295 Within Groups1213.512101.125 Total8963.7515