Download The effects of UVA on Vitamin D Treated Cells

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
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Radiates from the sun.
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Most radiation is stopped by the ozone layer
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Have shorter wavelengths than visible light, thus
are more powerful
Waves range from 100nm to 400nm
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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.
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Gram positive bacteria.
Common surface symbiont in many mammals
(including humans).
Most forms considered non-pathogenic.
Potentially pathogenic
Forms biofilms
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A group of fat-soluble secosteroids.
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The body can synthesize it with adequate sun exposure.
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Effects of supplementation are uncertain.
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Needed for bone growth.
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Liquid vitamin D is measured in IUs, which is the
measurement of concentration. 4,000 IUs per mL.
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Also called hypervitaminosis D.
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Results from excess vitamin D supplements.
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Can cause liver or kidney conditions.
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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.
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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
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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
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.
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.
248
245
0
5
10
20
TIME OF EXPOSURE (MIN)
237
251
P-Value=0.309
NUMBER OF CELL COLONIES
261
UVA STRESSED CELLS
30
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
6. 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
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
P-Value (Whole Graph=9.05239E-56)
Interaction=0.0029
P-Value=0.00037
249
Number of Cell Colonies
282
P-Value=0.848
285
249
257
256
P-Value=0.651
153
171
175
P-Value=9.952E-05
96
54
0S
2S
5S
10 S
UVC Exposure Time (sec)
0% D
1% D
45
10% D
P-Value=0.554
9
12
20 UVC
10
T-Crit = 1.94
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
10 UVC, 10% Vitamin D 1
Significant
Insignificant
P-Value=9.05239E-56
350
LD50= 5UVC
LD50= 6UVC
LD50=5.5UVC
Number of Colonies
300
250
200
0%
150
1%
100
10%
50
0
0 UVC
2 UVC
5 UVC
10 UVC
Exposure time (sec)
20 UVC
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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.
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UVA radiation was not strong enough
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UVA exposures weren’t long enough
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Only 6 replicates
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Only 4 exposure times
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Only 1 wavelength used (UVC 250nm)
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Plating may not have been synchronized
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Cannot analyze the health or growth rate of cells that
recovered from radiation
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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
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/