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

2. 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

3. 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)

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

5. 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

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

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

8. 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

9. 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 D ,000 IU)
Micropipettes
Sterile tips
Klett spectrophotometer
Labeling tape
Lab Conoco UVC hood (254nm
UVC cm2 at working
surface)

10. 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.

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

12. 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.

13. Vitamin D Remediation Effects
Vitamin D P-Value:
UV Light P-Value:
3.42E-0.6
Interaction:

14. 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

15. 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

16. Works Cited
“Vitamin D.” Vitamin D and UV Exposure | American Academy of Dermatology,
“Vitamin D and UV.” Science Learning Hub,
“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,
Lucas, Jim. “What Is Ultraviolet Light?” LiveScience, Purch, 15 Sept. 2017,
LD, Megan Ware RDN. “Vitamin D: Health Benefits, Facts, and Research.” Medical News Today, MediLexicon International, 13 Nov. 2017,

17. 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

18. Two Factor ANOVA ANOVA Source of Variation SS df MS F P-value F crit
Sample 2
Columns
3.24E-21
Interaction 4
Within
45762 36
Total 44

19. 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 2
Within Groups 12
2593.6
Total 14

20. 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 2
3.42E-06
Within Groups 12
Total 14

21. 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 2
Within Groups 12
912.8
Total 14

22. 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 2
9596.6
1.75E-05
Within Groups
3685.2 12
307.1
Total 14

23. 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 2
1.68E-08
Within Groups 12
1109.8
Total 14

24. 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 2
5.06E-08
Within Groups
9009.2 12
Total 14

25. Staph E. Survivorship