Daniel Love Central Catholic High School Grade 11

Caused by X-Rays and UV Rays Stress causes an increase in free radical production Cell degeneration possible Other effects include an increased risk of cancer or death

 Radiates from the sun.  Most radiation is stopped by the ozone layer  Have shorter wavelengths than visible light, thus are more powerful  Waves range from 100nm to 400nm

 In humans, causes sunburn, nausea, sun stroke and possibly skin cancer.  FDA protection methods include sunscreen, hats, sunglasses, and antiradiation clothing.  Can possibly cause dimers in a cell’s DNA, which leads to replication errors and mutations.

 Gram positive bacteria.  Common surface symbiont in many mammals (including humans).  Most forms considered non-pathogenic.  Potentially pathogenic  Forms biofilms

 A group of fat-soluble secosteroids.  The body can synthesize it with adequate sun exposure.  Effects of supplementation are uncertain.  Needed for bone growth.  Liquid vitamin D is measured in IUs, which is the measurement of concentration. 4,000 IUs per mL.

 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, known as Hypercalcemia

The purpose of this experiment is to determine whether vitamin D will significantly remediate the effects of UV radiation on S. epidermidis

Null Hypothesis- Vitamin D will have no significant effect on the survivorship of UV stressed Staph. Alternate Hypothesis- Vitamin D will have a significant effect on the survivorship of UV stressed Staph.

 LB agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride)  Staphylococcous epidermidis  Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)  Sterile test tubes  Sterile spreader bars  Incubator  Ethanol  Bunsen burner  Vortex  Vitamin D (liquid supplement)  Micropipettes  Sterile Tips  Klett Spectrophotometer  Labeling tape  Labconco UVC Hood (254nm UVC cm 2 at working surface)  UVA 50 watt lamp

1. Bacteria (Staph) was 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 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. 4. The cell concentration was then diluted to 10³ cells/mL mL of the cell concentration was added to the agar plate and exposed to UVA light at varying times. 6. The plates were incubated at 37°C overnight. 7. The resulting cell colonies were counted the next day. Each colony was assumed to have risen from one cell.

P-Value=0.309

1. Bacteria (Staph) was 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 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. 4. Concentrations of Vitamin D were made in separate tubes with concentrations of 0% (control), 1%, and 10%. 5. The cell concentration was then diluted and added to each tube. The cells were exposed to the vitamin D for ten minutes mL was then plated from each tube. 7. The cells were then exposed to timed amounts of UVC radiation (0s, 2s, 5s, 10s, and 20s) 8. The cells were incubated at 37°C overnight. 9. The resulting cell colonies were counted the next day. All colonies were assumed to have risen from one cell

Concentration0% (Control)1%10% S. epidermidis0.1mLs SDF9.9mLs9.8mLs8.9mLs Vitamin D0mLs0.1mLs1mL Final Volume10mLs

P-Value (Whole Graph= E-56) P-Value= P-Value=0.848 P-Value=0.651 P-Value=9.952E-05 P-Value=0.554

ConcentrationT-ValueSignificance 0 UVC, 1% Vitamin D0.54Insignificant 0 UVC, 10% Vitamin D4.33Significant 10 UVC, 1% Vitamin D 4.64Significant 10 UVC, 10% Vitamin D1Insignificant T-Crit = 1.94

P-Value= E-56 LD50= 5UVC LD50= 6UVC LD50=5.5UVC

 The null hypothesis was rejected for concentrations of 1% Vitamin D with a 10 second Exposure.  Null Hypothesis can be accepted for all other concentrations  1% Vitamin D was able to significantly remediate the UVC radiation.  UVA is much weaker than UVC and has a higher kill time.

 UVA radiation was not strong enough  UVA exposures weren’t long enough  Only 6 replicates  Only 4 exposure times  Only 1 wavelength used (UVC 250nm)  Plating may not have been synchronized  Cannot analyze the health or growth rate of cells that recovered from radiation

 More replicates and concentrations  More wavelengths  Longer exposure times for UVA in order to generate a kill curve  Use UVB instead of UVA  Conduct an agar infusion test to simulate longer exposure