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

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

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

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

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

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

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

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

9. 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

10. 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 UVC cm2 at working surface)

11. 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.

12. 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

13. S. epidermidis Survivorship

14. Vitamin D Remediation Effects

15. Dunnett’s Test
T-Crit = 1.98

16. 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

17. 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

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

19. References