UV Light Effects on Vitamin D Stressed Staph Cells Adam Parrish Central Catholic High School

Oxidative Stresses Caused by UV rays Increase in free radical production Increased risk of cancer Cell degeneration

Ultra Violet Light Radiation from the sun Most stopped by the ozone layer Shorter wavelength than visible=more powerful 100nm-380nm

Ultra Violet Radiation Effects Humans-sunburn, skin cancer, sun stroke FDA approved protection – sunscreen, hats, sunglasses, anti-radiation clothing

Vitamin D Fat-soluble secosteroids Bone growth Easily synthesized by the body Uncertain effects Measured in IUs, about 4,000 IUs per mL

Toxicity of Vitamin D Hypervitaminosis D Excess Vitamin D Liver and kidney damage Hypercalcemia – buildup of calcium in the bloodstream

Staphylococcus epidermidis Gram positive Non-pathogenic Common surface symbiont Forms biofilms

Purpose The purpose of this experiment is to determine if vitamin D will significantly improve the survivorship of UV stressed S. epidermidis.

Hypotheses Null Hypothesis – the Vitamin D will have no significant effect on the survivorship of UV stressed staph cells Alternate Hypothesis – the Vitamin D will have a significant effect on the survivorship of UV stressed staph cells

Materials Sterile Dilution Fluid [SDF] (200mM KH2O4, 100nM K2HPO4, 10mM MgSO4, 10nM MgSO4, 1mM NaCl) Sterile test tubes Sterile spreader bars Staphylococcus epidermidis Incubator Ethanol Bunsen burner Vortex LB agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Vitamin D (liquid supplement) Micropipettes Sterile tips Klett spectrophotometer Labeling tape Lab Conoco UVC hood (254nm UVC0.7-0.9 cm2 at working surface)

Procedure Bacteria (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. Concentration of Vitamin D were made in separate tube with concentrations of 0% (control), 1% and 10%. The cell concentration was then diluted and added to each tube. The cells were exposed to the vitamin D for 10 minutes. 0.1mL aliquots were then plated from each tube. The cells were exposed to UVC radiation at timed intervals of 0s, 2s, 5s, 10s, and 20s. The cells were incubated at 37°C overnight The resulting cell colonies were counted the following day. Colonies assumed to have risen from one cell.

Concentration chart Concentration 0% 1% 10% S. epidermidis 0.1 mL SDF 9.9 mL 9.8 mL 8.9 mL Vitamin D 0 mL 1 mL Final Volume 10 mL

S. epidermidis Survivorship

Vitamin D Remediation Effects

Dunnett’s Test T-Crit = 1.98

Conclusions The null hypothesis was rejected for the concentration of 1% Vitamin D with a 10 second exposure as well as 10% with a 0 second exposure The null hypothesis was accepted for all other concentrations 1% Vitamin D was able to significantly remediate UVC radiation

Limitations Only 6 replicates 4 exposure times 1 wavelength, (250nm) Plating may not have been synchronized Cannot analyze health or growth rate of cells that recovered

Extensions More replicates and concentrations More wavelengths Conduct an agar infusion test to allow for a longer exposure time

References http://hps.org/hpspublications/articles/uv.html http://earthobservatory.nasa.gov/Features/UVB/ http://www.who.int/uv/faq/whatisuv/en/index2.html http://www.who.int/uv/uv_and_health/en/