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

11. Procedure
6. 0.1mL of the cell cultures were added to the solutions which brought them to a total volume of 10mL.
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.
7. Solutions were pipetted onto LB plates using a micro-pipette.
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.
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.
4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL.
10. Colonies were counted, data was recorded, results were analyzed.
5. Solutions were made up in sterile test tubes using sterile fluid and variable.

12. Concentration in Tubes
Control (0%) 1%
10%
50%
Sterile Fluid
9.9 mL
9.8
8.9 mL
5 mL
Variable
0.0 mL
0.1 mL
1 mL
4.9 mL
E.Coli
Total Volume
10 mL

13. Statistical Analysis Dunnett's Test Anova: Single Factor
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
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

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. Gatorade Casein Protein
Dunnett's Test
Variable
Concentration
T-Value
Significance
About Time
Whey Protein 1%
10%
50%
3.62
5.43
7.88
Significant
Gatorade Casein Protein
5.24
5.77
Not Significant
GNC Brand Creatine
3.98
6.1
8.9

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
The Alternate 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. Creatine Anova

24. Works Cited http://www.webmd.com/men/creatine#1
protein
And a very special thanks to my mentor Mr. Krotec