Vitamin D Effects on UV-Stressed Microbes Robert Kania Central Catholic High School Grade 9

Ultra Violet Light Electromagnetic radiation Radiation from the sun Most stopped by the ozone layer Wavelength from 10nm to 400nm Produces about 10% of total light output from the sun

Ultra Violet Radiation Effects Sunburn, skin cancer, sun stroke Microflora effects? Common FDA approved protection – sunscreen, hats, sunglasses, anti-radiation clothing Produces many negative effects on life (free radicals)

Vitamin D Fat-soluble secosteroids Bone growth Easily synthesized by the body Uncertain effects Powerful antioxidant Can neutralize free radicals

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

Staphylococcus Epidermidis (Staph) Gram-positive bacterium Non-pathogenic Common surface symbiont Forms biofilms

Purpose To determine if Vitamin D will significantly improve the survivorship of UV-stressed Staphylococcus epidermidis.

Hypotheses Null Hypothesis – Vitamin D will not have a significant effect on UV- stressed Staph survivorship Alternate Hypothesis – Vitamin D will significantly increase survivorship of UV stressed Staph

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 (CARLSON® VITAMIN D3 - 10,000 IU) Micropipettes Sterile tips Klett spectrophotometer Labeling tape Lab Conoco UVC hood (254nm UVC 0.7-0.9cm2 at working surface)

Procedure 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), 0.1% and 1% 5. The cell concentration was then diluted with SDF and added to each tube. The cells were exposed to the vitamin D for 10 minutes.

Concentration Chart Concentration [0%] 0.1% 1% Staph E. 0.1 mL SDF Vitamin D 0 mL 0.01 mL Final Volume 10 mL

Procedure 6. 0.1mL aliquots were then plated from each tube. 7. The cells were exposed to UVC radiation at timed intervals of 0s, 4s, and 8s. 8. The cells were incubated at 37°C overnight 9. The resulting cell colonies were counted the following day. Colonies assumed to have risen from one cell.

Vitamin D Remediation Effects Vitamin D P-Value: 0.603121 UV Light P-Value: 3.42E-0.6 Interaction: 0.03509

Conclusions Vitamin D alone did not have a significant effect on Staph survivorship UV light significantly reduced survivorship It appeared Vitamin D had a significant remediation effect on Staph survivorship

Limitations and Extensions Only 5 replicates Only 3 exposure times Only one wavelength (250nm) Plating may not have been synchronized Only survivorship assessed More replicates More concentrations of Vitamin D Different wavelengths of UV light Other bacterial models Other antioxidants Other cell stresses

Works Cited “Vitamin D.” Vitamin D and UV Exposure | American Academy of Dermatology, www.aad.org/media/stats/prevention-and-care/vitamin-d-and-uv-exposure. “Vitamin D and UV.” Science Learning Hub, www.sciencelearn.org.nz/resources/1313-vitamin-d-and-uv. “Ultraviolet Radiation: How It Affects Life on Earth : Feature Articles.” NASA, NASA, earthobservatory.nasa.gov/Features/UVB/. Otto, Michael. “Staphylococcus Epidermidis – the ‘Accidental’ Pathogen.” Nature Reviews. Microbiology, U.S. National Library of Medicine, Aug. 2009, www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/. Lucas, Jim. “What Is Ultraviolet Light?” LiveScience, Purch, 15 Sept. 2017, www.livescience.com/50326-what-is-ultraviolet-light.html. LD, Megan Ware RDN. “Vitamin D: Health Benefits, Facts, and Research.” Medical News Today, MediLexicon International, 13 Nov. 2017, www.medicalnewstoday.com/articles/161618.php.

Data Chart 0 sec 4 sec 8 sec 0% 252 292 11 348 200 21 220 36 216 196 13 366 236 17 0.10% 284 308 44 260 336 35 32 328 304 28 344 364 1% 272 288 123 300 79 320 316 70 102 332 310 136

Two Factor ANOVA ANOVA Source of Variation SS df MS F P-value F crit Sample 28892.13 2 14446.07 11.36442 0.000149 3.259446 Columns 583469.7 291734.9 229.5017 3.24E-21 Interaction 14769.33 4 3692.333 2.904681 0.03509 2.633532 Within 45762 36 1271.167 Total 672893.2 44  

Single Factor First Column Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 1402 280.4 5124.8 Column 2 1564 312.8 1515.2 Column 3 1512 302.4 1140.8 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 2736.533 2 1368.267 0.527555 0.603121 3.885294 Within Groups 31123.2 12 2593.6 Total 33859.73 14  

Single Factor First Row Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 1402 280.4 5124.8 Column 2 1176 235.2 1571.2 Column 3 98 19.6 98.8 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 194238.4 2 97119.2 42.8795 3.42E-06 3.885294 Within Groups 27179.2 12 2264.933 Total 221417.6 14  

Single Factor 2nd Column Anova: Single Factor SUMMARY Groups Count Sum Average Variance 5 1176 235.2 1571.2 0.1 1604 320.8 843.2 1 1550 310 324 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 21731.73 2 10865.87 11.90389 0.001417 3.885294 Within Groups 10953.6 12 912.8 Total 32685.33 14  

Single Factor 3rd Column Anova: Single Factor SUMMARY Groups Count Sum Average Variance 5 98 19.6 98.8 0.1 175 35 1 510 102 787.5 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 19193.2 2 9596.6 31.2491 1.75E-05 3.885294 Within Groups 3685.2 12 307.1 Total 22878.4 14  

Single Factor 2nd Row Anova: Single Factor SUMMARY Groups Count Sum Average Variance 0 sec 5 1474 294.8 2451.2 4 sec 1604 320.8 843.2 8 sec 175 35 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 249756.1 2 124878.1 112.523 1.68E-08 3.885294 Within Groups 13317.6 12 1109.8 Total 263073.7 14  

Single Factor 3rd Row Anova: Single Factor SUMMARY Groups Count Sum Average Variance 0 sec 5 1512 302.4 1140.8 4 sec 1550 310 324 8 sec 510 102 787.5 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 139136.5 2 69568.27 92.66297 5.06E-08 3.885294 Within Groups 9009.2 12 750.7667 Total 148145.7 14  

Staph E. Survivorship