The Effects of Supplements on Prokaryotic Survivorship

1. The Effects of Supplements on Prokaryotic Survivorship Luke Nedrow Grade 11 Central Catholic High School

2. Background Information • The industry for performance enhancing supplements is worth approximately 16 billion dollars • The supplement industry is not regulated by the FDA or any other company • This results in these companies cutting health for drugs and chemical combinations that boost performance • Potential dangers are: kidney functions, calcium loss, protein, heavy metal contamination, and the effect of unknown chemical combinations • With all these potential dangers and lack of regulation, the results can be harming

3. Casein Protein Casein is the name for a family of related phosphoproteins The four main types of casein proteins make up 80% of proteins in mammalian milk and 40% in human milk Casein protein tends to give your blood stream and steady supply of amino acids for hours after consumption Also casein proteins are often found in cheeses, paint, glue, and plastics

4. Whey Protein Whey protein typically comes in four major forms: concentrate, isolate, hydrolysate, and Native Whey Whey protein is a mixture of proteins isolated from whey, created as a by-product of cheese production The protein in mammalian milk is 20% whey protein and 60% in human milk Various studies have concluded that a steady diet helps fight against specific cancers About Time Protein is all natural, gluten free, lactose free, and non-GMO

5. Creatine Supplement Creatine is a nitrogenous organic acid that is found in vertebrates Its main role is to facilitate recycling of adenosine triphosphate, the energy currency of the cell, primarily in muscle and brain tissue Creatine is naturally produced in the human body from the amino acids glycine and arginine Dietary supplementation with creatine may improve muscle wasting associated with cancer and other chronic diseases.

6. Escherichia coli  Large group of gram negative bacteria One of the most common forms of bacteria Commonly found in the digestive tracts of animals Major cause of food borne illnesses Is one of the most popular models for prokaryotic cells

7. Human Micro Flora • Little is known about the association between humans and their flora • Some of the effects are mutualistic parasitic, pathogenic, and commensal • About 10% of the biomass on the body are symbionts • E. Coli is one of the most prominent and well known models • E. Coli was chosen for this experiment because of it’s diversity • It lives in the intestinal tract of humans and many other mammals • It is a common prokaryotic cell model

8. Purpose • To determine the effects of different types of supplements on E. coli survivorship.

9. Hypothesis Null Hypothesis- The supplements will not significantly effect the survivorship of E. coli. Alternate Hypothesis- The supplements will have a significant effect on the survivorship of E. coli. Alternate Hypothesis- The creatine will have the most significant effect on the Survivorship of E. Coli

10. Materials • E.coli • Muscle Milk Whey protein shake • Gatorade Casein Protein shake • About Time Whey Protein shake • GNC brand creatine shake • LB media (.5% yeast extract, 1% tryptone, 1% sodium chloride) • Agar plates • Spreader bars • Bunsen Burner • Vortex • Incubator • Micropipettes • Sterile pipette tips • Sterile Dilution Fluid • Turntable • Sterile test tubes • Test tube rack • Ethanol • Matches • Sidearm flask • Labeling tape

11. 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 dilution fluid to a concentration of approximately 10^5 cells/mL. 5. Solutions were made up in sterile test tubes using sterile fluid and variable. 6. 0.1mL of the cell cultures were added to the solutions which brought them to a total volume of 10mL. 7. Solutions were pipetted onto LB plates using a micro-pipette. 8. Spreader bars were sterilized and used to spread solutions on the plates. 9. Plates were put in an incubator set at 37 degrees Celsius for 48 hours. 10. Colonies were counted, data was recorded, results were analyzed.

12. Concentration in Tubes

13. Statistical Analysis Anova: Single Factor Dunnett's Test Statistical test that compares an experimental group directly to the control group Identifies that the experimental group is significantly different than the control if t- value > t-crit • Analysis of variation • Statistical test that compares the means of multiple groups • P-value from ANOVA testing identifies significance if P-value is < 0.05

14. About Time Brand Whey Protein P-Value: 1.2^ -11 Cell Count Averages

15. Gatorade Brand Casein Protein P-Value:1.78 ^ -9 Cell Count Averages

16. GNC Brand Creatine P-Value:1.42 ^ -13 Cell Count Averages

17. Dunnett'sTest

18. Conclusion • The Null Hypothesis that states that the supplements will not significantly effect the survivorship of E. coli can be rejected because the p-value for each supplement was above .05 • TheAlternate Hypothesis that states that the supplements will have a significant effect on the survivorship of E. coli can be accepted because the p-value for each supplement was above .05 • Alternate Hypothesis that states that the creatine will have the most significant effect on the survivorship of E.coli can be accepted because in each of the three concentrations the Creatine had the highest t-value

19. Limitations • Plating was not perfectly synchronized • Error in plating and pipetting techniques are possible • LB plates do not properly represent muscle cells • Inability to count the exact amount of cells on each plate • Limited number of low concentrations • Only one exposure time • Only survivorship was tested

20. Extensions • Test longer exposure times (especially for casein proteins) • Test different brands of protein • Test against not just E.Coli but Staph as well

21. Whey Protein Anova

22. Casein Protein Anova

23. CreatineAnova

24. Works Cited • http://www.webmd.com/men/creatine#1 • https://en.wikipedia.org/wiki/Whey_protein • http://www.chemistryislife.com/the-chemistry-of-whey-protein • http://www.mensfitness.com/nutrition/supplements/5-benefits-of-casein-protein • And a very special thanks to my mentor Mr. Krotec