The Effects of Sports Drinks on Microbial Flora

Patrick MarchioneCentral Catholic High School

Grade 9

First sports drink created by a chemist, William Owen, in the U.K. in 1927.

In 1968, sports drinks began to revolutionize sports.

Used for rapid replacement of electrolytes and fluid lost during exercise.

Restores depleted muscle carbohydrate storages that cause muscle failure and fatigue.

Sports Drinks

Water Glucose Sucrose Dextrose Total Carbohydrates 34g Citric acid Natural flavor Sodium citrate Monopotassium phosphate Gum arabic Red 40 Glycerol ester of rosin

Gatorade Ingredients Protein 0g Sodium 270 mg Potassium 75mg Total Fat 0g

Vitamin Water Ingredients Reverse osmosis water Crystalline fructose Cane sugar Taurine 25 mg Total Carbohydrate 32g Total Fat 0g Sodium 0g Protein 0g Less than 0.5% of:

Vitamin C Vitamin B12 Natural flavors Vegetable juice Magnesium lactate

Chromium polynicotinate Citric acid Vitamin B6 Calcium lactate Potassium phosphate Vitamin B5 Zinc gluconate

Micro Bacterial Flora Present in the respiratory, urinary, and digestive tracts as well as on

skin.

Consists of mostly bacteria, few protists and fungi.

Most are harmless, but some can cause diseases.

Influences human anatomy, physiology, and overall health.

1014 bacteria are harbored within and on humans.

Many products are tested for effects on human cells. Micro Flora Effects?

Gram positive coccus

Common surface symbiont in many mammals

(including humans)

Most forms considered non-pathogenic.

Potentially pathogenic upon systemic entry

Forms biofilms

Staphylococcus epidermidis

Rod-shaped, 2 micrometers diameter

Gram negative bacteria

Reproduction rate: 20 min

Survival, growth, and replication require only a single carbon

source and ammonium salts.

Common symbiont found in the lower intestines of warm blooded

animals.

Few pathogenic - Almost 73,000 cases of infection and 61 deaths

per year in the United States.

Common prokaryote model

Escherichia coli

To determine the effects of Gatorade and Vitamin Water on S. epidermidis and E. coli survivorship.

Purpose

Null HypothesisGatorade and Vitamin Water will not have a

significant effect on the survivorship of E. coli and S. epidermidis

Alternate HypothesisGatorade and Vitamin Water will significantly

alter the survivorship of E. coli and S. epidermidis

Materials• Gatorade sports drink• Vitamin Water sports drink• Mirco pipettes + Sterile tips• Spreader bars• 60 LB agar plates(1% Tryptone,

0.5% Yeast extract, 1% NaCl) • Escherichia coli Dh5- alpha (E.

Coli) (Obtained from Doonan Lab, CMU)

• Staphylococcus espidermidis (Staph) (Obtained from Ward’s Scientific)

• Burner• Sidearm Flask• Vortex• Klett Spectrophotometer• SDF (Sterile Dilution Fluid)

(10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)

• 8 sterile test tubes• Ethanol

1. Bacteria (E. coli and Staph) were grown overnight in sterile LB media.

2. Samples of the overnight culture were added to fresh media in a sterile sidearm flask.

3. The cultures were placed in incubators at 37°C until a density of 50 Klett spectrophotometer units were reached.

4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10E5 cells/mL.

5. Various concentrations of two different sports drinks (0%, 1%, 10%, and 50%) were created in test tubes containing SDF, resulting in 10 mL per tube

Procedure (Liquid Exposure)

Concentration ChartE. coli/Staph 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Variable 0 mL 0.1 mL 1 mL 5 mL

SDF 9.9 mL 9.8 mL 8.9 mL 4.9 mL

Concentration

[0%] [1%] [10%] [50%]

6. 100 µL of cell culture was then added to the test tubes, yielding a final volume of 10 mL.

7. The solutions were mixed by vortexing and allowed to sit at room temperature for 15 minutes.

8. After vortexing to evenly suspend cells,100 µL were removed from the tubes and spread on LB plates.

9. The plates were incubated at 37°C for 24 hours.10. The resulting colonies were counted. Each colony is assumed to

have arisen from one cell.

Procedure (Liquid Exposure continued)

AnovaStatistical test that allows the comparison of different groups (between group variation vs. within group variation)

Dunnett’s testA follow up statisical test for the comparison of a particular variable group vs. the control group to determine significant variation

Anova and Dunnett’s Test

Sport Drink Effects on E. coli Survivorship

0% 1% 10% 50%0

100

200

300

400

500

600

700

638.375 637

597.75

468

638.375

573.5

528.5

427

Gatorade

Vitamin Water

Concentration of Variable (Gatorade/Vitamin Water)

Nu

mb

er

of

Su

rviv

ing

Co

lon

ies

P value 0.000134

P value 0.00017

Sport drink effects on S. epidermidis Survivorship

0% 1% 10% 50%0

20

40

60

80

100

120

108.875

95.75

77.5

69.5

108.875

90.75

80.576.5

Gatorade

Vitamin Water

Concentration of Variable (Gatorade/Vitamin Water)

Nu

mb

er

of

Su

rviv

ing

Co

lon

ies

P value 0.068637

P value 1.7E-05

T-crit= 3.29

Dunnett’s Test E. coli Gatorade Concentration

T value Significance

[1%] 0.97 Not Significant

[10%] 2.24 Not Significant

[50%] 6.42 Significant

T-crit= 3.29

Dunnett’s Test E. coli Continued..Vitamin Water Concentration

T value Significance

[1%] 1.33 Not Significant

[10%] 2.98 Not Significant

[50%] 6.71 Significant

T-crit= 3.29

Dunnett’s Test S. epidermidisGatorade Concentration

T value Significance

[1%] 4.36 Significant

[10%] 6.93 Significant

[50%] 8.06 Significant

The null hypothesis can be accepted for: All concentrations of Vitamin Water [E. coli]

1%, 10% of Gatorade [E. coli]

1%, 10% of Vitamin Water [Staph]

The null hypothesis can be rejected for: All concentrations of Gatorade [Staph]

50% Vitamin Water [Staph]

50% Gatorade [E. coli]

Conclusions

Limitations

❖ Plating slightly unsynchronized

❖ Only 4 concentrations of variables tested

❖ Only one kind of test, Liquid Pulse

Extensions

❖ Use more types of sports drinks

❖ Use more concentrations

❖ Agar Infusion

❖ Synergistic effects

"Escherichia Coli." MicrobeWiki. Kenyon.edu, n.d. Web. 20 Dec. 2012.

Orenstein, Abigail. "The Discovery and Naming of Staphylococcus Aureus." Antimicrobe.org. N.p., n.d. Web. 20 Dec. 2012.

Todar, Kenneth, PhD. "Staphylococcus Aureus." Staphylococcus Aureus. N.p., 2008. Web. 20 Dec. 2012.

"Sports Drinks: The Real Facts." Healthportal. N.p., n.d. Web. 28 Jan. 2015.

"Who Invented Sports Drinks? - HowStuffWorks." HowStuffWorks. N.p., n.d. Web. 28 Jan. 2015.

References

Anova- alpha 0.05Anova: Single Factor Vitamin Water E. coli

SUMMARYGroups Count Sum Average Variance

Column 1 4 2439 609.75 710.9167Column 2 4 2294 573.5 137Column 3 4 2114 528.5 217

Column 4 4 1708 427 4876.667

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 75102.69 3 25034.23 16.85357 0.000134 3.490295Within Groups 17824.75 12 1485.396

Total 92927.44 15

Anova- alpha 0.05Anova: Single Factor Gatorade E. coli

SUMMARY

Groups Count Sum Average Variance

Column 1 4 2668 667 1162

Column 2 4 2548 637 556

Column 3 4 2391 597.75 1154.917

Column 4 4 1872 468 4805.333

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 92233.19 3 30744.4 16.01636 0.00017 3.490295

Within Groups 23034.75 12 1919.563

Total 115267.9 15

Anova- alpha 0.05Anova: Single Factor Vitamin Water Staph

SUMMARY

Groups Count Sum Average Variance

Column 1 4 364 91 86.66667

Column 2 4 363 90.75 57.58333

Column 3 4 322 80.5 86.33333

Column 4 4 306 76.5 49

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 644.6875 3 214.8958 3.074516 0.068637 3.490295

Within Groups 838.75 12 69.89583

Total 1483.438 15

Anova- alpha 0.05Anova: Single Factor Gatorade Staph

SUMMARY

Groups Count Sum Average Variance

Column 1 4 507 126.75 128.9167

Column 2 4 383 95.75 73.58333

Column 3 4 310 77.5 67

Column 4 4 278 69.5 135

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 7750.25 3 2583.417 25.54677 1.7E-05 3.490295

Within Groups 1213.5 12 101.125

Total 8963.75 15