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

Gatorade 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 Protein 0g 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

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

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

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

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

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

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

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.

Materials 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 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

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.

Concentration Chart Gatorade Salt Solution SDF Bacteria (Staph) 0 mL 9.9 mL 0.1 mL 0% G 0% S 0.5 mL 9.4 mL 5% G 0% S 2 mL 7.9 mL 20% G 0% S 9.8 mL 0% G 1% S 0% G 5% S 9.3 mL 5% G 1% S 8.9 mL 5 % G 5% S 7.8 mL 20% G 1% S 7.4 mL 20% G 5% S

Concentrations simplified Gatorade Salt Low (5%) High (20%) 0 0 0 L 0 H Low (1%) L 0 L L L H High (5%) H 0 H L H H

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.

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.

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

P Value: 0.0192

P Value: 0.02145

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.

Limitations & Extensions 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

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

Survivorship ANOVA

Biofilm ANOVA