Patrick Marchione Central Catholic High School Grade 9

 First sports drink created by a chemist, William Owen, in the U.K. in  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

 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

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

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.  bacteria are harbored within and on humans.  Many products are tested for effects on human cells. Micro Flora Effects?

 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

 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

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

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

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

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)

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%]

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)

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

Sport Drink Effects on E. coli Survivorship P value P value

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

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

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

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

 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

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

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

Anova- alpha 0.05 Anova: Single FactorVitamin Water E. coli SUMMARY GroupsCountSumAverageVariance Column Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups Within Groups Total

Anova- alpha 0.05 Anova: Single FactorGatorade E. coli SUMMARY GroupsCountSumAverageVariance Column Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups Within Groups Total

Anova- alpha 0.05 Anova: Single FactorVitamin Water Staph SUMMARY GroupsCountSumAverageVariance Column Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups Within Groups Total

Anova- alpha 0.05 Anova: Single FactorGatorade Staph SUMMARY GroupsCountSumAverageVariance Column Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups E Within Groups Total