The Effects of Sports Drinks on E.coli Survivorship Jonathan Farkos Grade 9 Pittsburgh Central Catholic High School

Problem Do sports drinks have adverse effects on microflora cells?

Human Symbionts, Pathogens, and Flora More than 10% of your body mass is due to symbionts and pathogens Mostly Prokaryotic cells Some may cause diseases Also protect the body from foreign invaders Reinforce immune system, prevent allergies, and produce vitamins Lie within skin, saliva, and gastrointestinal tracts Break down abnormally large nutrients

E.Coli c600 Used in many cell/biochemical investigations Easy to manipulate in labs Normally found in the intestines of animals. Some are pathogenic, but most are harmless Large and diverse group of gram(-) bacteria Utilized in this study as a human cell model and as a model of pathogens E.coli was chosen for this experiment because of it’s diversity, it lives in the intestinal tract of humans and many other mammals, and it serves as a common prokaryotic cell model.

Gatorade Very commonly used beverage amongst athletes and non- athletes Provides electrolytes Common uses include: During sports, Post-sport recovery, and as a common drink Thought to be non-toxic substance with no effects on body Ingredients: Contains water, sucrose (table sugar), dextrose, citric acid, natural flavor, sodium chloride (table salt), sodium citrate, monopotassium phosphate, and flavoring/coloring ingredients, and sucrose acetate isobutyrate.

To assess the effects of Gatorade on the survivorship of E.coli The human micro flora performs many functions vital  to human health. Purpose

Hypotheses Null Hypothesis: Gatorade will not affect the survivorship of E.coli Alternative Hypothesis: Gatorade will significantly reduce the survivorship of E.coli

Materials YEPD agar plates (1% Yeast Extract, 2% peptone, 2% dextrose) Sterile pipette tips Micropipettes Vortex Incubator Spreading turntable Spreader bar Sports Drink (Gatorade) Sterile capped test tubes with Sterile Dilution Fluid [SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, .1mM CaCl2, 100mM NaCl) E.Coli C600

Procedure 1.Bacteria (E.coli) were grown overnight in sterile LB media. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask.   3. The cultures were placed in an incubator (37˚C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8 cells/mL. 4. The cultures were diluted in sterile fluid to a concentration of approximately 10^5 cells/mL. 5: LB agar plates(1% tryptone, .5% yeast extract, 1%NaCl) were prepared 6: Gatorade was pipetted into tubes containing SDF, to create the following concentrations: 0%, 1%, 10%, 25%, and 50%, yielding a final volume of 10 mL.

Procedure (Continued) Concentration Chart 0% 1% 10% 25% 50% Sterile Dilution Fluid 9.9 mL 9.8 mL 8.9 mL 7.4 mL 4.9 mL Microbe 0.1 mL Gatorade 0 mL 1 mL 2.5 mL 5 mL Total Volume 10 mL CHANGE The cells were vortexed for even suspension. 8. After vortexing to evenly suspend cells, 0.1mL aliquots were removed from the tubes at time 10 minutes and 50 minutes and spread on YEPD agar plates (5 plates per concentration)

Procedure (Continued) The plates were then incubated at 30 degrees for 48 hours. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

P Value - 0.000528 P Value - Less than 1x10E-6

Variable Concentration Variable Concentration Dunnett’s Test T-Critical = 3.48 10 Min Exposure Variable Concentration T-Value Interpretation 1% Gatorade 1.12 Not Significant 10% Gatorade 2.09 25% Gatorade 0.54 50% Gatorade 5.42 Significant 50 Min Exposure Variable Concentration T-Value Interpretation 1% Gatorade 0.46 Not Significant 10% Gatorade 10.73 Significant 25% Gatorade 6.83 50% Gatorade 14.36 Significant change. Because anovas suggested that there was significant difference within varaition or groups. DON’T SAY PROVE significant variance from control CONFIDENCE

Analysis of Exposure Time Did the duration of exposure have a significant effect? Ratio of Survivorship of Variables to Control (10 Min Exposure) 1% Gatorade 10% Gatorade 25% Gatorade 50% Gatorade Control (0% Gatorade) 96% 93% 102% 83% Ratios of Survivorship of Variables to Control (50 Min Exposure) 1% Gatorade 10% Gatorade 25% Gatorade 50% Gatorade Control (0% Gatorade) 101% 71% 81% 61% Key Phrases: the less you say about stats the better it is. Increasing percentage survirorship was decreaed ANOVA anaylsis was performed to see im th emean within these groups varied significantly. The analysis revealed significance suggested that 2 of the means varied significantly. Because ANOVA showed significant variation exsts you want to find out of any variable group varied significantly from the control. Dunnettts were performed to compare the variables back to the control to check for significance. Alpha is 95% confidence. Don’t state too much. Questions about stats. T-test is a restricted test to two variables of an ANOVA.

Conclusions Generally, increased concentrations of Gatorade lowered the amount of E.coli cells that grew while some concentrations provided aid to growth Increased exposure did have a significant effect on the amount of surviving colonies. Higher concentrations of Gatorade could also be effective in reducing the amount of E.coli colonies that could survive

Limitations Limited number of concentrations Only 1 species test Only 2 exposure times During the testing, plating could have possibly been unsynchronized. Only one type of Sports Drink was used.

Further Studies Different types of Sports Drinks More of a variety of concentrations Experiment on other types of cells Don’t read off of the screen. Become more confident.

Sources https://www.cdc.gov/ecoli/general/ http://www.webmd.com/a-to-z-guides/tc/e-coli-infection-topic-overview#1 https://microbewiki.kenyon.edu/index.php/Escherichia_coli http://www.healthline.com/health/food-nutrition/is-gatorade-bad-for- you#1 https://sites.psu.edu/siowfa15/2015/09/15/the-science-behind-gatorade/ Don’t use first person throughout the presentation. D

Amount of Colonies per plate (10 min Exposure) Data Results Amount of Colonies per plate (10 min Exposure) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Average 0% Gatorade 300 284 297 355 326 312.4 1% Gatorade 293 298 306 311 301.2 10% Gatorade 302 286 280 291.6 25% Gatorade 348 310 307 303 321 317.8 50% Gatorade 235 238 279 261 258.6

Data Results (Continued) Amount of Colonies per plate (50 min Exposure) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Average 0% Gatorade 369 383 346 355 362 363 1% Gatorade 390 361 372 354 367.6 10% Gatorade 244 266 253 261 258 256.4 25% Gatorade 319 295 293 287 282 295.2 50% Gatorade 228 215 231 217 211 220.4

Survivorship of E.coli Scatterplot arithmic graph Change colors.

Data Analysis (ANOVA)