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 nonathletes 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.
PURPOSE To assess the effects of Gatorade on the survivorship of E.coli The human micro flora performs many functions vital to human health.
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) Sterile Dilution Fluid Concentration Chart 0% 1% 10% 25% 50% 9.9 mlchange 9.8 ml 8.9 ml 7.4 ml 4.9 ml Microbe 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml Gatorade 0 ml 0.1 ml 1 ml 2.5 ml 5 ml Total Volume 10 ml 10 ml 10 ml 10 ml 10 ml 7. 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) 9. The plates were then incubated at 30 degrees for 48 hours. 10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.
Colonies 450 400 350 300 250 200 150 100 50 0 The Effects of Sports Drinks on E.coli Survivorship P Value - 0.000528 P Value - Less than 1x10E-6 0% 1% 10% 25% 50% Concentrations (10 Minutes) (50 Minutes)
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 Not Significant 25% Gatorade 0.54 Not Significant 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 Significant 50% Gatorade 14.36 Significant
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%
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
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-foryou#1 https://sites.psu.edu/siowfa15/2015/09/15/the-science-behind-gatorade/
DATA RESULTS 0% Gatorade 1% Gatorade 10% Gatorade 25% Gatorade 50% Gatorade Amount of Colonies per plate (10 min Exposure) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Average 300 284 297 355 326 312.4 293 298 306 311 298 301.2 302 293 286 297 280 291.6 348 310 307 303 321 317.8 280 235 238 279 261 258.6
DATA RESULTS (CONTINUED) 0% Gatorade 1% Gatorade 10% Gatorade 25% Gatorade 50% Gatorade Amount of Colonies per plate (50 min Exposure) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Average 369 383 346 355 362 363 390 361 361 372 354 367.6 244 266 253 261 258 256.4 319 295 293 287 282 295.2 228 215 231 217 211 220.4
Colonies SURVIVORSHIP OF E.COLI 450 400 350 300 250 200 150 100 50 0 The Effects of Sports Drinks on E.coli 0% 1% 10% 25% 50% Concentrations (10 Minutes) (50 Minutes)
DATA ANALYSIS (ANOVA)