Synergistic Effects of Vitamin B12 and Creatine on Microbes Jake Rocchi CCHS, 11th grade 2nd year in PJAS
Vitamin B12 Water-soluble vitamin Needed for proper functioning in brain, nervous system, and blood formation Unable to be produced by animals, plants, and fungi Recommended amount per day: 1000 mcg/1 ml
Creatine Monohydrate Nitrogenous organic acid Synthesized in liver and kidney Supplies energy to muscles by increasing production of ATP 95% is stored in the skeletal muscles Many athletes take Creatine supplements to increase strength and hypertrophy Recommended amount per day: 5000 mg (5 g)
Microbial Flora Refers to microorganisms living in or on the body Little is known about the association between humans and their flora Effects are mutualistic, parasitic, pathogenic, and commensal Perform functions beneficial to the host, including the manufacture of essential vitamins, and the prevention of colonization by undesirable microbes Human foods and supplements may have unintended effects on the flora populations and their functions
Gram Positive vs Gram Negative Bacteria Most pathogenic bacteria in humans are Gram (+) organisms. Simple cell wall Some antibiotics work against the formation of the cell wall Cell contains an extra layer of lipopolysaccharides for extra protection Outer membrane protects bacteria from several antibiotics
Escherichia coli (E. coli) Gram-Negative Bacteria Common bacteria found in various organisms in the ecosystem Most forms are harmless, but some can become pathogens
Staphylococcus epidermidis Common surface symbiont in many mammals Gram positive bacteria Most strains considered nonpathogenic. Pathogenic strains can be lifethreatening
Purpose To determine the individual effects of creatine monohydrate and Vitamin B12 on E. coli and Staph cell survivorship To determine the synergistic effects of creatine monohydrate and Vitamin B12 on E. coli and Staph cell survivorship
Null/Alternative Hypothesis Null: Creatine monohydrate and Vitamin B12 will not have a significant effect on E. coli and Staph survivorship, either individually or synergistically. 1. Creatine monohydrate and Vitamin B12 will each have negative effects on E. coli and Staph survivorship. 2. Synergistic effects of the two substances will reduce E. coli and Staph survivorship.
Materials GNC Creatine Monohydrate 5000 GNC Liquid Vitamin B12 (cherry) Micro pipettes + Sterile tips Spreader bars 72 LB agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl) Escherichia coli DH5-alpha (E.Coli) (Obtained from Doonan Lab, CMU) Staphylococcus epidermidis (Staph) (Obtained from Ward s Scientific) Burner Sidearm Flask Turn-table Vortex Incubator Gloves/goggles Klett Spectrophotometer SDF (Sterile Dilution Fluid) Sterile Test Tubes (10 mm KH2PO4, 10 mm K2HPO4, 1 mm MgSO4, 0.1 mm CaCl2, 100 mm NaCl) Ethanol
Procedure (Liquid Pulse) 1. Bacteria (E.coli and Staph) 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. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/ml. 5. Creatine monohydrate and Vitamin B12 were sterile filtered 6. Variables and bacteria were added to SDF to generate the desired concentrations:
Procedure (cont.) 0% Creatine 0.1x Creatine x Creatine 0% Vitamin B12 9.9 ml SDF 0 ml Creatine 0 ml Vitamin B12 0.1 ml bacteria Total: 10 ml 9.8 ml SDF 0.1 ml Creatine 0 ml Vitamin B12 0.1 ml bacteria Total: 10 ml 8.9 ml SDF 1.0 ml Creatine 0 ml Vitamin B12 0.1 ml bacteria Total: 10 ml 0.1x Vitamin B12 9.89 ml SDF 0 ml Creatine 0.01 ml Vitamin B12 0.1 ml bacteria Total: 10 ml 9.79 ml SDF 0.1 ml Creatine 0.01 ml Vitamin B12 0.1 ml bacteria Total: 10 ml 8.89 ml SDF 1.0 ml Creatine 0.01 ml Vitamin B12 0.1 ml bacteria Total: 10 ml x Vitamin B12 9.8 ml SDF 0 ml Creatine 0.1 ml Vitamin B12 0.1 ml bacteria Total: 10 ml 9.7 ml SDF 0.1 ml Creatine 0.1 ml Vitamin B12 0.1 ml bacteria Total: 10 ml 8.8 ml SDF 1.0 ml Creatine 0.1 ml Vitamin B12 0.1 ml bacteria Total: 10 ml Note: Creatine was made from 10x stock Vitamin B12 was made from 100x stock
Procedure (cont.) 6. 0.1 ml of cell culture was then added to the test tubes, yielding a final volume of 10 ml. 7. The solution was mixed by vortexing and allowed to sit at room temperature for 15 minutes. 8. After vortexing to evenly suspend cells, 0.1 ml were removed from the tubes and spread on LB agar plates. 9. The plates were incubated at 37 C for 24 hours. 10. The resulting colonies were counted. Each colony is assumed to have risen from one cell.
ANOVA Analysis of Variation Statistical test that compares the means of multiple groups P-value from ANOVA testing identifies significance if < 0.05
Creatine Effects on E. coli Survivorship Number of Colonies 320 240 160 80 P value: 3.42E-06 0 Control 0.1xC xc Concentrations Vitamin B12 Effects on E. coli Survivorship Number of Colonies 320 240 160 80 0 Control 0.1xB12 xb12 P value: 0.028304 Concentrations
Vitamin B12 and Creatine Effects on E. coli Survivorship 360 P value: 2.32376E-12 270 Number of Colonies 180 90 0 Control 0.1xB12 0.1xB12 xb12 xb12 0.1xC xc 0.1xC xc Concentrations
Creatine Effects on Staph Survivorship Number of Colonies 400 300 200 100 P value: 1.21E-10 0 Control 0.1xC xc Concentrations Vitamin B12 Effects on Staph Survivorship Number of Colonies 330 247.5 165 82.5 P value: 0.009011 0 Control 0.1xB12 xb12 Concentrations
Vitamin B12 and Creatine Effects on Staph Survivorship 400 P value: 2.6443E-11 300 Number of Colonies 200 100 0 Control 0.1xB12 0.1xB12 xb12 xb12 0.1xC xc 0.1xC xc Concentrations
Conclusions The null hypothesis should be rejected. Creatine significantly decreased the survivorship of both E. coli and Staph. Vitamin B12 decreased the survivorship of E. coli but caused an increase of survivorship in Staph. Synergistically, creatine and vitamin B12 decreased E. coli and Staph survivorship (except for the x Creatine + 0.1x Vitamin B12 solution for E. coli).
Limitations and Extensions Limitations Time of spread-plating slightly varied Only 2 concentrations of each variable used Only one type and time of exposure (liquid pulse) Only survivorship assessed Extensions Test other microbial models Use more concentrations Agar infusion Use different forms of the variables Growth curve assessment
References http://www.jissn.com/content/4/1/6 http://www2.centralcatholichs.com/biology/ https://umm.edu/health/medical/altmed/supplement/creatine http://www.nlm.nih.gov/medlineplus/ency/article/002403.htm http://en.m.wikipedia.org/wiki/staphylococcus_epidermidis http://en.m.wikipedia.org/wiki/escherichia_coli
2 Factor ANOVA (E. coli) Source of Variation SS d f MS F P-value F crit Sample 2832.888889 2 1416.444444 6.01486258 0.00691149 3.354130829 Columns 26727.72222 2 13363.86111 56.74898754 2.1373E-10 3.354130829 Interaction 49556.77778 4 12389.19444 52.61011285 2.32376E-12 2.727765306 Within 6358.25 2 7 235.4907407 Total 85475.63889 3 5
Two Factor ANOVA (Staph) Source of Variation SS df MS F P-value F crit Sample 17801.38889 2 8900.694444 65.16235087 4.64144E-11 3.354130829 Columns 374154.0556 2 187077.0278 1369.598631 7.21088E-28 3.354130829 Interaction 23350.44444 4 5837.611111 42.73739154 2.6443E-11 2.727765306 Within 3688 27 136.5925926 Total 418993.8889 35