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 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

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 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

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 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

14. 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

15. Procedure (cont.)
8. The solutions were vortexed and allowed to sit at room temperature for 10 minutes.
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)
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.
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
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:

19. P Value:

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
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
*Dr. Carrie Doonan, CMU, for lab space and equiptment

24. Survivorship ANOVA

25. Biofilm ANOVA