Daniel Love Central Catholic High School Grade 11.

The Effects of UVA on Vitamin D Treated

Cells Daniel LoveCentral Catholic High SchoolGrade 11

• Caused by X-Rays and UV Rays• Stress causes an increase in free radicalproduction• Cell degeneration possible• Other effects include an increased risk ofcancer or death

Oxidative Stress

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

Ultra Violet Radiation

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 errorsand mutations.

Effects of UV Radiation

Gram positive bacteria.

Common surface symbiont in many mammals (including humans).

Most forms considered non-pathogenic. Potentially pathogenic Forms biofilms

Staphylococcus epidermidis

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.

Vitamin D

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

Vitamin D Toxicity

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

Purpose

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.

Hypotheses

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 UVC0.7-0.9 cm2 at working surface)

UVA 50 watt lamp

Materials

1. Bacteria (Staph) was grown overnight in sterile LBMedia.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.5. 0.1mL 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.

Procedure UVA

0 5 10 20 30

261

251

248

245

237

UVA Stressed Cells

Time of Exposure (min)

Num

ber

of

Cell

Colo

nie

sUVA Effects on S. epidermidis

P-Value=0.309

1. Bacteria (Staph) was grown overnight in sterile LBMedia.2. A sample of the overnight culture was added to fresh media ina sterile sidearm flask.3. The culture was placed in an incubator (37°C) until a densityof 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 minutes6. 0.1mL 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

Procedure UVC

Concentration 0% (Control)

1% 10%

S. epidermidis 0.1mLs 0.1mLs 0.1mLs

SDF 9.9mLs 9.8mLs 8.9mLs

Vitamin D 0mLs 0.1mLs 1mL

Final Volume 10mLs 10mLs 10mLs

Concentration chart

0 s 2 s 5 s 10 s 20 UVC

282

249

153

54

9

285

257

171

96

12

249 256

175

45

10

Interaction=0.0029

0% D 1% D 10% D

UVC Exposure Time (sec)

Num

ber

of

Cell C

olo

nie

s

Vitamin D UVC Remediation Effects

P-Value (Whole Graph=9.05239E-56)

P-Value=0.00037P-Value=0.848

P-Value=0.651

P-Value=9.952E-05

P-Value=0.554

Concentration T-Value Significance

0 UVC, 1% Vitamin D 0.54 Insignificant

0 UVC, 10% Vitamin D 4.33 Significant

10 UVC, 1% Vitamin D

4.64 Significant

10 UVC, 10% Vitamin D

1 Insignificant

Dunnett’s TestT-Crit = 1.94

0 UVC 2 UVC 5 UVC 10 UVC 20 UVC0

50

100

150

200

250

300

350

0%1%10%

Exposure time (sec)

Num

ber

of

Colo

nie

sS. epidermidis SurvivorshipP-Value=9.05239E-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.

Conclusions

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

Limitations

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

Extensions

http://www.epa.gov/sunwise/doc/uvradiation.html

http://hps.org/hpspublications/articles/uv.html

http://earthobservatory.nasa.gov/Features/UVB/

http://www.skincancer.org/prevention/uva-and-uvb

http://www.who.int/uv/faq/whatisuv/en/index2.html

http://www.who.int/uv/uv_and_health/en/

References