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Creatine Effects on Microbial Flora Andrew Haky 11 th Grade Pittsburgh Central Catholic HS.

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Presentation on theme: "Creatine Effects on Microbial Flora Andrew Haky 11 th Grade Pittsburgh Central Catholic HS."— Presentation transcript:

1 Creatine Effects on Microbial Flora Andrew Haky 11 th Grade Pittsburgh Central Catholic HS

2 Microbial Flora The skin has a normal flora of eukaryotic fungi, protists, and bacteria Not much is known about the association between humans and their flora Effects can be mutualistic, parasitic, pathogenic, and commensal Normal Flora provide nutritional and digestive benefits, secrete vitamins, stimulate antibody production, and protect against pathogenic microbes Supplements taken by humans might have unintended effects on these important functions of the flora Bacteria capable of expressing specific enzymes to degrade creatine have been found in the bacterial flora of the human colon

3 Creatine An organic acid naturally synthesized from amino acids (methionine, glycine, arginine) primarily in the kidney and liver, then transported in the blood for use by muscles. Approximately 95% located in the skeletal muscle. Direct relation to ATP production and storage. For an adult 70 kg male, daily turnover of creatine has been estimated at about 2g.

4 Supplemental Creatine Most popular bodybuilding supplement on the market. Has such an impact because it super-hydrates muscle cells with water. Enhances muscle growth, and strengthens fibers. Increases energy levels, strength, and recovery rates. Accelerates weight loss and builds lean body mass. Used to increase athletic performance, in the treatment of heart disease, muscular dystrophy, and Parkinson’s Disease.

5 Two Types of Creatine: Free Creatine (Crf) – One-third of skeletal muscle is present in Crf form. Phosphorylated (Crphos) – The rest is present in Crphos form.

6 Creatine Increases Phosphocreatine storage Increases ability of muscles to resynthesize ATP from ADP Thus, increased energy demands can be met. “Studies have shown that both Crf and Crphos levels in skeletal muscle can be increased, and performance of high intensity intermittent exercise enhanced, following a period of creatine supplementation.”

7 Previous Studies: Effects on Isokinetic Muscle Torque: – “muscle peak torque production was greater in all subjects, when compared with the data prior to creatine ingestion.” – Placebo effect showed no difference Increase in Creatine pool: – “supplementation with 5g of creatine monohydrate resulted in a significant increase in the total creatine content of the quadriceps femoris muscle measured in 17 subjects.”

8 Escherichia coli (E. coli) Cells are rod shaped and usually about 2 micrometers in length Gram-negative Part of the human flora; found in the human colon and digestive tract Reproduces rapidly, often within thirty minutes Commonly used as a prokaryote cell model Many strains, most non-pathogenic Free living, symbiotic or pathogenic

9 Staphylococcus epidermidis Part of our normal skin flora, gram-positive Anaerobic, but grows best in aerobic conditions Opportunistic pathogen, requires major breach in hosts defenses Non-pathogenic strain utilized

10 Purpose To determine the effects of creatine on E.coli and Staph e. survivorship/proliferation

11 Hypothesis Creatine will significantly increase Staph. and E. coli proliferation/ survivorship. NULL Hypothesis Creatine will not significantly affect Staph or E. coli proliferation/ survivorship. Microbes may degrade creatine into by-products that can benefit cell metabolism.

12 Materials  Ethanol (sterilization of instruments)  Latex gloves  E. coli DH5 alpha  Staphylococcus epidermidis  Micropipette  Tube rack  Test Tubes  SDF (per 1 liter) (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl)  LB agar plates  LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)  Bunsen burner  Spreader bars  Matches  Sterile pipette tips  Incubator  Vortex  Klett spectrophotometer and sidearm flasks  Creatine

13 Procedure 1)E. coli and Staphylococcus epidermidis were grown overnight in sterile LB media. 2)A sample of the overnight 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 10 8 cells/mL. 4)The culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/mL. 5) The tubes were prepared as follows:

14 Table of Concentrations LIQUID EXPOSURE 0% Concentration 0.1% Concentration 1% Concentration 10 % Concentration Microbe E.coli/Staph 0.1mL SDF 9.9mL9.89mL9.8mL8.9mL Creatine Solution 0mL0.01mL0.1mL1mL Final Volume 10mL 5 replicates per group

15 Procedure, con’t 6) The E. coli and Staphylococcus epidermidis were exposed to the creatine concentrations for 20 minutes. 7)100 µL aliquots were removed from the tubes and spread on LB plates.(5 replicates) 8)The plates were incubated at 37 degrees Celsius for 24 hours. 9)The resulting colonies were quantified. Each colony is assumed to have arisen from one cell.

16 P-value = 0.000251

17 P-value=9.33E-06

18 Dunnet’s Test Test GroupT-valueInterpretation e.coli groups: 0.1% creatine3.51Significant 1% creatine3.00Insignificant 10% creatine5.91Significant staph. groups 0.1% creatine0.56Insignificant 1% creatine3.20Insignificant 10% creatine7.05Significant T-crit value=3.29

19 Analysis of Results Did liquid exposure to creatine significantly affect E.coli and Staph. proliferation/survivorship? – P-value= 0.000251 (E.coli), SIGNIFICANT – P-value=9.33E-06 (Staph.), SIGNIFICANT Which concentrations of creatine significantly effected e. coli proliferation/survivorship? – 0.1% creatine, T-value: 3.51, SIGNIFICANT – 1% creatine, T-value: 3.00, INSIGNIFICANT – 10% creatine, T-value: 5.91, SIGNIFICANT Which concentrations of creatine significantly effected staph. proliferation/survivorship? – 0.1% creatine, T-value: 0.56, INSIGNIFICANT – 1% creatine, T-value:3.20, INSIGNIFICANT – 10% creatine, T-value:7.05, SIGNIFICANT

20 Conclusion The null hypothesis can be rejected because the variable did have a significant effect. It appears that creatine did have a positive survivorship effect on the microbial flora tested. There are many reasons why creatine could have had this positive survivorship effect. However, microbial creatine degradation at present is not completely understood. An evolutionary screening and characterization of enzymes involved in digestion is necessary for this understanding.

21 Limits: Only one exposure time was used Ways to better dissolve creatine More replicates Better synchronization of plating A growth curve analysis Other microbial models Synergistic effects of multiple variables Creatine effects on stressed microbes Could test creatine on a model representing muscle cells Creatine effects on stem cell differentiation Other variables: whey, glutamine Extensions:

22 Sources Balsom PD, Söderlund K, and Ekblom B. "Creatine in Humans with Special Reference to Creatine Supplementation." National Center for Biotechnology Information. US National Library of Medicine, 18 Oct. 1994. Web. 12 Nov. 2011.. "Creatine Background : What Is Creatine?" Creatine Information Center. Web. 12 Nov. 2011.. "Creatine." Wikipedia, the Free Encyclopedia. Web. 12 Nov. 2011.. Greenhaff PL, Hultman E, Soderlund K, Harris R, Short AH, and Casey A. "Influence of Oral Creatine Supplementation of Muscle Torque during Repeated Bouts of Maximal Voluntary Exercise in Man." Department of Physiology and Pharmacology, Queens Medical Centre, University of Nottingham, U.K., May 1993. Web. 12 Nov. 2011.. Harris RC, Söderlund K, and Hultman E. "Elevation of Creatine in Resting and Exercised Muscle of Normal Subjects by Creatine Supplementation." Department of Clinical Chemistry II, Karolinska Institute, Huddinge University Hospital, Sweden, Sept. 1992. Web. 12 Nov. 2011.. Http://, Ron Jonk-. "Creatine." University of Maryland Medical Center | Home. Web. 12 Nov. 2011.. Hu, Amanda. "Looks Can Be Deceiving: the Case of Escherichia Coli." Journal of Young Investigators 6.5 (2002). Web. 12 Nov. 2011.. "Staphylococcus Epidermidis." Medpedia. Web. 12 Nov. 2011..

23 E.coli Analysis of Variance Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column 15625125109 Column 25793158.6161.8 Column 35769153.8192.2 Column 45908181.6453.8 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups8108.5532702.8511.792540.0002513.238872 Within Groups3667.216229.2 Total11775.7519

24 Staph. Analysis of Variance Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column 15652130.446.8 Column 25677135.4368.3 Column 35796159.2318.7 Column 45969193.874.7 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups12560.234186.73320.713589.33E-063.238872 Within Groups323416202.125 Total15794.219

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