Vitamin D Effects on Microbial Flora Justin Beiriger Central Catholic High School Grade 10 2nd Year in PJAS

Microbial Flora Studies in the past have been done on human cells. Scientist are finding out that microbial flora that live in the body are just as important. This experiment is being conducted on microbial flora.

Vitamin D A group of fat-soluble secosteroids. Nicknamed “the sunshine vitamin”. Because the body can synthesize it with adequate sun exposure. Effects of supplementation are uncertain. Needed for bone growth. Can cause a build-up of calcium.

Vitamin D Toxicity Also called hypervitaminosis D. Results from excess vitamin D supplements. Can cause liver or kidney conditions. Main consequence is a build-up of calcium in the bloodstream. Called hypercalcemia

Chemistry of Vitamin D Ultraviolet light produces the vitamin. Binds to a protein transcription factor. Regulates gene expression. Outcome is the maintenance of calcium and phosphorus levels in the bones and blood.

Previous Studies Edward Mellanby first discovered vitamin D. 1918-1920 He wrote extensively on vitamin deficiency. Previous studies of vitamin D supplementation have produced inconsistent results, with some trials showing a decrease in inflammatory markers and others showing no effect.

E. coli Large and diverse group of gram (-) bacteria Surrounded by an extra cell wall composed of lipopolysaccharides. Free living, symbiotes, or pathogens. Most strains are not pathogenic. Serves as a common prokaryotic cell model

Staphylococcous epidermidis Human skin flora. Gram (+) bacteria. Surrounded by a simple cell wall. Most forms are non-pathogenic. Forms biofilms on plastic devices.

Purpose To determine what effect vitamin D has on E. coli and Staph survivorship.

Hypotheses Null hypothesis: Vitamin D will not significantly affect the survivorship of E. coli or Staph. Alternative hypothesis: Vitamin D will significantly reduce the survivorship of E. coli and/or Staph.

Materials LB agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Bunsen burner Vortex Vitamin D (liquid supplement) Escherichia coli (DH5-alpha) Staphylococcous epidermidis Micropipettes Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) Klett Spectrophotometer Turntable Labeling tape Micro rack Sterile test tubes Micro tubes Sterile spreader bars 0.22 micron sterile filter Incubator Ethanol

Procedure Bacteria (E. coli and Staph) was grown overnight in sterile LB media. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108 cells/mL. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL. The Vitamin D was sterile filtered through a 0.22 micron syringe filter. Vitamin D was mixed with the appropriate amounts of SDF to create vitamin D concentrations of 10%, 1%, and 0.1%.

Table of Concentrations 0% Vitamin D 0.1% Vitamin D 1% 10% Vitamin D Microbe 0.1 mL SDF 9.9 mL 9.89 mL 9.8 mL 8.9 mL 0 mL 0.01 mL 1 mL Total 10 mL

Procedure 100 µL aliquots of cell culture was then added to the vitamin D solutions, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL. The solutions were vortexed and allowed to sit at room temperature for a 20 minute incubation period. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB plates. The plates were incubated at 37˚C for 24 hours. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

Liquid Exposure Table (Staph) 0% Vitamin D 0.1 % Vitamin D 1% 10% 154 182 140 106 160 179 137 103 159 172 131 92 174 187 134 97 163 194 144 88 166 176 136 148 184 127 180

Liquid Exposure Graph (Staph) P-Value = 1.43E-15 Significant

Dunnett’s Test (Staph) T-Critical = 2.88 Alpha = 0.05 Concentration of Vitamin D T-Value Significant? 0.1% 5.79 Significant 1% 6.99 10% 15.50

Liquid Exposure Table (E. coli) 0% 0.1% 1% 10% 139 189 96 77 148 176 102 82 137 177 113 79 142 168 109 70 153 184 92 86 136 140 106 75 130 180 103 147 169 110

Liquid Exposure Graph (E. coli) P-Value = 1.01E-15 Significant

Dunnett’s Test (E. coli) T-Critical = 2.88 Alpha = .05 Concentration of Vitamin D T-Value Significant? 0.1% 6.46 Significant 1% 7.65 10% 12.05

Comparison of Survivorship (Staph and E. Coli)

Conclusion The alternate hypothesis can be accepted. The null hypothesis can be rejected. In small quantities, the vitamin D promoted bacterial growth. For both Staph and E. coli. In excess, the bacteria were killed by the vitamin D.

Limitations The plating was not perfectly synchronized. Some cells could have had longer or shorter exposure times. The liquid vitamin D was removed from pill form, so it possibly contained excess pieces of the capsule.

Extensions More trials to create a better basis for evaluating the results Prolonged exposure test using vitamin D infused into the agar Using an antibiotic resistant strain of bacteria for the test model

References http://ods.od.nih.gov/factsheets/vitamind/ http://www.medicalnewstoday.com/articles/161618.php http://www.webmd.com/vitamins-supplements/ingredientmono-929-VITAMIN%20D.aspx?activeIngredientId=929&activeIngredientName=VITAMIN%20D http://www.mayoclinic.com/health/vitamin-d/NS_patient-vitamind http://www.webmd.com/a-to-z-guides/e-coli-infection-topic-overview http://www.medicalnewstoday.com/articles/68511.php http://kidshealth.org/kid/stay_healthy/food/ecoli.html http://www.about-ecoli.com/ http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html http://microbewiki.kenyon.edu/index.php/Staphylococcus_epidermidis http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1001133 http://wiki.medpedia.com/Staphylococcus_epidermidis