The Effects of Gatorade and Salinity on Biofilms

1. The Effects of Gatorade and Salinity on Biofilms Renne Cabacungan Grade 11 Central Catholic High School

2. Gatorade • Used for rapid replacement of electrolytes and fluid lost during exercise. • Restores depleted muscle carbohydrate storages that cause muscle failure and fatigue.

3. 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

4. Morton’s Table Salt • Serving size: ¼ cup; 560 mg Sodium • Solar Evaporation Extraction & Mine Production • Iodized salt • Salinity and cellular hypertonicity

5. Biofilms • Coherent and generally adherent cells • Extracellular Polymeric Substance/Extracellular Matrix • Phenotypic shift in gene regulation • Lateral gene transfer

6. Biofilm Inhibition • Adherence- conditioning films, polysaccharides • Anti-biofilm agents: chemical inhibitors to adherence and activity • Anti-microbial surface conditioning • Hydrophobic • Nontoxic biofilm agents

7. Staphylococcus Epidermidis • Bacteria usually non-pathogenic towards humans • Commonly found on the epidermis of humans • Common prokaryote model • Gram + bacteria

8. Rationale/Application • Biofilms account for 80% of infectious disease • Developing information on biofilm formation/inhibition • Past studies of Gatorade/Salinity antimicrobial effects • Medical implications: • Biofilm targeted disinfectants • Potential for salinity treatments

9. Purpose • To determine the effects of Gatorade on biofilm formation • To determine the effects of salt on biofilm formation • To test for an interactive effect amongst the two variables • To test for relationship with antimicrobial effects

10. Hypothesis • Null Hypothesis: Salt and Gatorade will not have a significant effect on Staph epidermidis biofilm formation and survivorship. • Alternative Hypothesis: Salt and Gatorade alone will have a significant effect on Staph epidermidis biofilm formation and survivorship. • Salt and Gatorade together will have an interactive effect on biofilm formation and survivorship.

11. Materials • Staph. E culture (Ward’s Science) • Gatorade • Table Salt • LB agar plates • LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) • Sterile dilution fluid (100 mM KH2PO4, 100 mM K2HPO4, 10 mM MgSO4, 1mM NaCl) • Sterile pipette tips • Micropipettes • Vortex • Incubator (37 C) • Sidearm Flask • Sterile Spreader Bars • Ethanol • 96 well tissue culture treated microtiter dish • Crystal Violet • Acetic Acid • Microtiter plate absorbance reader

12. Survivorship Procedure • 1. Staph. E was grown overnight in sterile LB Media. • 2. The culture was 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 10⁸ cells/mL. • 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL. • 5. A solution of salt and SDF were created as a 20% stock. • 6. The Gatorade and salt water were sterilized by means of a 0.2 micron syringe filter • 7. Experimental variables were mixed with the appropriate amounts of SDF to create concentrations.

13. Concentration Chart

14. Concentrations simplified

15. Procedure (cont.) • 8. The solutions were vortexed and allowed to sit at room temperature for 10 minutes. • 9. 100 µL aliquots were removed from the tubes and spread on LB-agar plates. • 10. The plates were incubated at 37°C for 48 hours. • 11. The resulting colonies were counted visually (each colony was assumed to have arisen from one cell) • 12. Steps were repeated for 7 replicates.

16. Biofilm Formation • Growing a Biofilm • 1. Tubes were prepared according to the concentration chart (scaled down to microtube volume of 2mL) • 2. 200 µL from the tubes was added per well in a 96 well dish. 8 replicates were performed from each tube. • 3. The microtiter plate was incubated for 48 hours at 37°C. • Staining the Biofilm • 1. After incubation, the cells were gently removed out by turning the plate and allowing to drip dry. • 2. The plate was gently submerged in a small tub of water. The plate was allowed to drip dry. • 3. 200 µL of a 0.1% solution of crystal violet in water was added to each well of the microtiter plate.

17. Biofilm Formation (cont.) • 4. The microtiter plate was incubated at room temperature for 10 minutes. • 5. The plate was rinsed by submerging in a tub of water as outlined above. • 6. The microtiter plate was turned upside down and dried overnight. • Quantifying the Biofilm • 1. 200 µL of 30% acetic acid in water was added to each well of the microtiter plate to solubilize the CV. • 2. The microtiter plate was incubated at room temperature for 10 minutes. • plate reader at 550 nm using 30% acetic acid in water as the blank. • 3. The absorbance of the microtiter plates was quantified in a microtiter

18. P Value: 0.0192

19. P Value: 0.02145

21. Conclusions • Survivorship: The data supports the rejection of the null hypothesis, only in the presence of high salt and Gatorade content, possibly implying a synergistic effect. • Biofilm: The data supports the rejection of the null hypothesis, only in the presence of high salt and Gatorade content, possibly implying a synergistic effect. • In lower concentrations, the null hypothesis is accepted, the data does not support a significant effect in both cases, for lower concentrations.

22. Limitations & Extensions • Limitations: • Only one exposure time • Technique Errors • Only Inhibition/Survivorship tested • Limited Replicates • Only Tested One Model Organism • Reliability of biofilm assay • Extensions: • More Replicates • Multiple Model organisms • Test Reproduction/Promotion • More Concentrations • Different Types of Salt/Different Sports Drinks

23. Sources • https://www.future-science.com/doi/full/10.4155/fmc.15.7 • http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146668 • https://www.livestrong.com/article/43161-list-ingredients-gatorade/ • http://www.mortonsalt.com/article/morton-iodized-table-salt-nutritional-facts/ • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732559/ • https://www.ncbi.nlm.nih.gov/pubmed/23635385 • *Dr. Carrie Doonan, CMU, for lab space and equiptment

24. Survivorship ANOVA

25. Biofilm ANOVA