Transcript CCHS JonathanKrystopolski

VITAMIN E REMEDIATION
OF UV STRESSED STAPH
JONATHAN KRYSTOPOLSKI-CZERNICS
VITAMIN E
• Vitamin E - fat soluble nutrient.
• - Known as tocopherol
• - Acts as an antioxidant
• - Protects cells from free radicals.
ULTRAVIOLET LIGHT
• Ultraviolet light - form of radiation not visible to our eyes
• - Invisible on the electromagnetic spectrum
• - Can damage living tissues by creating free radicals
• - UV wavelength used was 254nm, germicidal
FREE RADICALS
Free radicals
- Unstable molecules created by chemical changes in the body
- - Can harm living tissues and can be created with UV light.
- - Can be fought with antioxidants in the immune system.
- - Might be neutralized by antioxidants
Microbial Flora
• • Little is known about the association between humans and their
• flora
• • Effects are mutualistic, parasitic, pathogenic and commensal
• • Provide nutritional and digestive benefits, secrete vitamins,
• stimulate antibody production, and protect against pathogenic
• microbes
Staph
WHAT IS STAPH
• Staphylococcus epidermidis
• - bacteria found on the skin.
• - Can cause multiple infections.
• - gram positive
• - easy to grow in lab setting
• - can produce biofilms
PURPOSE
This experiment was conducted to test the
remedial effects of vitamin E on UV stressed
staph.
The question that was asked was:
“Can Vitamin E protect Staph cells from
ultraviolet light?”
HYPOTHESIS
• Null Hypothesis: Vitamin E will not appear to have a
remedial effect on UV stressed staph.
• Alternative Hypothesis: Vitamin E will have a remedial
effect on UV stressed Staph.
SIMILAR EXPERIMENTS
• An experiment was conducted by Wen-Hsing
Cheng, Beth A. Valentine , and Xin Gen Lei
• They tested the ability of vitamin E to replace
Cellular Glutathione Peroxidase(GPX1)
• They observed that there was no evidence to say
that vitamin E was able to replace GPX1
MATERIALS
• Liquid vitamin E
Sterile Dilution Fluid [SDF} (100mM
KH2PO4, 100mM K2HPO4, 10mM
• Staphylococcus epidermidis
MgSO4, 1mM NaCl)
• Micropipette
UV culture hood
• Sterile Spreader bars
• Ethel alcohol
• Bunsen burner
• Sterile Micropipette tips
Incubator
15ml conical tubes
• LB agar (0.5% yeast extract, Vortex
1% tryptone, 1% sodium
Sterile water
chloride)
• Plates
Sharpie
PROCEDURE
1. The bacteria was grown until a density of 50 klett
spectrophotometer density was reached. This was approximately
10^8 cells/mL.
2. All equipment was sterilized.
3. The desired amount of amount of liquid vitamin E was
pipetted into each conical that contained sterile fluid.
4. 0.1ml of staph was pipetted into the each of the conical tubes.
5. The mixture was incubated for 10 minutes at room
temperature while being rolled back and forth in order to mix the
solution with constant inversion.
TABLE OF CONCENTRATIONS
Materials In Tubes
Control Group
Concentration 1
Concentration 2
Sterile Fluid
9.9ml
9.8 ml
8.9 ml
Vitamin E
0ml
.1ml
1ml
Staph
.1ml
.1ml
.1 ml
Total
10ml
10ml
10ml
PROCEDURES CONTINUED
• 6. .1ml of solutions was pipetted onto LB auger
• 7. the plates were exposed to uv light for selected intervals.
• 8. The plates were incubated for 1 day and the colonies
were counted (the colonies were recounted after a 48 hour
period).
VITAMIN E EFFECTS ON UV
STRESSED STAPH
300
Vitamin E Interaction
P Value: .616
250
150
100
Number of Colonies
200
239.2
220.4
UV light Interaction
P value:3e-06
Two Factor Interaction
P Value: 2.92
201
50
16
17.8
26.4
15.8
5.6
0
1
UV Exposure
Times
2
3 Seconds
0 Seconds
0 ml Vitamin E
.1 ml Vitamin E
3
6 Seconds
1ml Vitamin E
19
DUNNETT'S TEST ANALYSIS
Dunnet's Test formula:
T=m1-mc/square root of 2mse/n
Variable
T-crit
T Value
Interpretation
0ml vit E
3 seconds UV
3.90
8.05
Significant
0ml vit E
6 seconds UV
3.90
8.43
Significant
CONCLUSION
Hypothesis : The null hypothesis could not be
rejected
- Vitamin E did not appear to have a remedial
effect on UV stressed staph.
LIMITATIONS
-Spread plating was not performed in perfect
synchronization, therefore some bacteria had a
slightly higher exposure time
-Only 3 different UV exposure times were tested
-Only 3 concentrations of vitamin E were tested
- Only one species
EXTENSIONS
• -Increase the number of replicates.
• -Alter concentration of vitamin
• -Test different vitamins and/or
models.
BIBLIOGRAPHY
• http://householdproducts.nlm.nih.gov/cgi-bin/household/brands?tbl=chem&id=2211
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/
• http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html
• https://ods.od.nih.gov/factsheets/VitaminE-Consumer/
• http://www.webmd.com/vitamins-supplements/ingredientmono-954vitamin%20e.aspx?activeingredientid=954&activeingredientname=vitamin%20e
• http://www.ncbi.nlm.nih.gov/pubmed/10364708
• http://www.livescience.com/50326-what-is-ultraviolet-light.html
• http://science.ksc.nasa.gov/mirrors/msfc/description/ultraviolet.html
• http://jn.nutrition.org/content/129/11/1951.short
SINGLE FACTOR ANOVA RESULTS
TWO FACTOR ANOVA RESULTS.