The Effects of Gatorade on Microbial Survivorship Christopher Samek Grade 9 Pittsburgh Central Catholic High School 1st Year in PJAS

Problem Gatorade is constantly consumed to rehydrate the body. At what concentrations does Gatorade affect the survivorship of human microflora?

Sports Drinks 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.

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

Microbial Flora Microorganisms that reside in or on the surface of the body Respiratory, urinary, digestive tracts, and skin Consists of mostly bacteria, fungus, protists, and archea Most are harmless, but some can cause disease Most studies based on ingested materials study human cells, but human are a collection of cells and symbiotants Estimated 10% body mass is symbiotants

Staphylococcus epidermidis Human skin flora Gram (+) bacterium Surrounded by a simple cell wall Most forms are non-pathogenic Forms biofilms on plastic devices

Escherichia coli Large and diverse group of gram (-) bacteria Surrounded by an extra cell wall composed of lipopolysaccharides Free living, symbiotes, or pathogens Most strains are non-pathogenic Serves as a common prokaryotic cell model

Purpose To determine what effect Gatorade has on Staph e. and E. coli survivorship

Hypotheses Null : Gatorade will not have a significant effect on the survivorship of E. coli or Staph. e. Alternate : Gatorade will have a significant effect on the survivorship of the E. coli and/or Staph. e.

Materials 64 LB Agar Plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Escherichia Coli (DH5-alpha) Staphylococcus Epidermidis Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) Sterile test tubes Racks Sterile spreader bars Matches Incubator Ethanol Bunsen burner Vortex Gatorade Micropipettes Turn table Labelling tape 0.22 Micron sterile filter

Procedure E. coli and Staph. e. were grown overnight in sterile LB media. Samples of the overnight culture were added to fresh media in a sterile sidearm flask. The cultures were incubated until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8 - 10^9 cells/mL. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL. The Gatorade was diluted with sterile dilution fluid to concentrations of 0%, 5%, 10%, and 25% to total 9.9 mL.

Table of Concentrations 0% Concentration 5% 10% Concentration 25% Concentration Sterile Water 9.9 mL 9.4 mL 8.9 mL 7.4 mL Gatorade 0 mL 0.5 mL 1 mL 2.5 mL Staph. e. / E. coli 0.1 mL Total Volume 10 mL

Procedure 0.1 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. The tubes were allowed to incubate at room temperature for 10 minutes. After vortexing to evenly suspend cells, 0.1 mL. aliquots were removed from the tubes and spread on LB agar plates. The plates incubated at 37°C overnight. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

Results P-Value = 0.49 Not Significant Surviving Colonies 0% Concentrations of Gatorade

Results Survivorship of Staph. e. when Exposed to P-Value = 1.10E-34 Significant Survivorship of Staph. e. when Exposed to Various Concentrations of Gatorade 0% 5% 10% 25%

Dunnett’s Test (Staph. e.) T-Critical – 2.88 Alpha – .05 Concentration of Gatorade T-Value Significant? 5% 8.93 Significant 10% 5.40 25% 11.12

Conclusion Staph. e. The null hypothesis can be rejected. The alternate hypothesis can be accepted. Gatorade promoted bacterial growth in small quantities. Gatorade killed the bacteria in larger quantities. E. coli The null hypothesis fails to be rejected. The alternate hypothesis can be rejected. Gatorade had no effect on the E. Coli in any amount.

Limitations / Extensions Plating not perfectly synchronized Slightly shorter or longer exposure time Only two species tested Extensions More trials to create a better basis for evaluating the results Vary exposure time in a two-factor experiment Use agar infused plates

References https://en.wikipedia.org/wiki/Gram-positive_bacteria https://www.gatorade.com https://www.cdc.gov/ecoli/general/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/ http://www.livestrong.com/article/43161-list-ingredients-gatorade/

Liquid Exposure Raw Data (E. coli) 0% 5% 10% 25% 85 80 71 89 81 96 88 82 91 84 79 74 77 90 76 86 87

Liquid Exposure Raw Data (Staph. e.) 0% 5% 10% 25% 392 569 302 203 406 582 317 198 421 579 298 187 394 566 319 196 412 304 199 399 578 301 184 414 589 297 197 411 572 294 201

E. coli Anova Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 8 678 84.75 19.07142857 Column 2 681 85.125 32.69642857 Column 3 653 81.625 39.41071429 Column 4 659 82.375 24.26785714 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 71.84375 3 23.94791667 0.829749936 0.488737273 2.946685266 Within Groups 808.125 28 28.86160714 Total 879.96875 31  

Staph. e. Anova Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 8 3249 406.125 105.5535714 Column 2 4601 575.125 68.125 Column 3 2432 304 84.57142857 Column 4 1565 195.625 44.55357143 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 625149.8438 3 208383.2813 2752.718936 1.09513E-34 2.946685266 Within Groups 2119.625 28 75.70089286 Total 627269.4688 31