Transcript Vit D effects on flora Justin Beiriger new PJAS Slides 2012

Vitamin D Effects on
Microbial Flora
Justin Beiriger
Central Catholic High School
Grade 10
2nd Year in PJAS
Microbial Flora
• Studies in the past have been done on human
cells.
• Scientist are finding out that microbial flora
that live in the body are just as important.
• This experiment is being conducted on
microbial flora.
Vitamin D
• A group of fat-soluble secosteroids.
• Nicknamed “the sunshine vitamin”.
– Because the body can synthesize it with adequate
sun exposure.
• Effects of supplementation are uncertain.
• Needed for bone growth.
– Can cause a build-up of calcium.
Vitamin D Toxicity
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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.
– Called hypercalcemia
Chemistry of Vitamin D
• Ultraviolet light produces the vitamin.
• Binds to a protein transcription factor.
– Regulates gene expression.
• Outcome is the maintenance of calcium and
phosphorus levels in the bones and blood.
Previous Studies
• Edward Mellanby first discovered vitamin D.
– 1918-1920
– He wrote extensively on vitamin deficiency.
• Previous studies of vitamin D supplementation
have produced inconsistent results, with some
trials showing a decrease in inflammatory
markers and others showing no effect.
E. coli
• Large and diverse group of gram (-) bacteria
– Surrounded by an extra cell wall composed of
lipopolysaccharides.
• Free living, symbiotes, or pathogens.
• Most strains are not pathogenic.
• Serves as a common prokaryotic cell model
Staphylococcous epidermidis
• Human skin flora.
• Gram (+) bacteria.
– Surrounded by a simple cell wall.
• Most forms are non-pathogenic.
• Forms biofilms on plastic devices.
Purpose
To determine what effect vitamin D
has on E. coli and Staph
survivorship.
Hypotheses
• Null hypothesis: Vitamin D will not
significantly affect the survivorship of
E. coli or Staph.
• Alternative hypothesis: Vitamin D
will significantly reduce the
survivorship of E. coli and/or Staph.
Materials
• LB agar plates (0.5% yeast
extract, 1% tryptone, 1%
sodium chloride)
• Escherichia coli (DH5-alpha)
• 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
• Klett Spectrophotometer
• Turntable
• Labeling tape
• Micro rack
• Micro tubes
• 0.22 micron sterile filter
Procedure
1. Bacteria (E. coli and 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 culture was diluted in sterile dilution fluid to a
concentration of approximately 105 cells/mL.
5. The Vitamin D was sterile filtered through a 0.22 micron
syringe filter. Vitamin D was mixed with the appropriate
amounts of SDF to create vitamin D concentrations of 10%,
1%, and 0.1%.
Table of Concentrations
0%
0.1%
1%
10%
Vitamin D Vitamin D Vitamin D Vitamin D
Microbe 0.1 mL
0.1 mL
0.1 mL
0.1 mL
SDF
9.9 mL
9.89 mL
9.8 mL
8.9 mL
Vitamin
D
0 mL
0.01 mL
0.1 mL
1 mL
Total
10 mL
10 mL
10 mL
10 mL
Procedure
6. 100 µL aliquots of cell culture was then added to the
vitamin D solutions, yielding a final volume of 10 mL and a
cell density of approximately 103 cells/mL.
7. The solutions were vortexed and allowed to sit at room
temperature for a 20 minute incubation period.
8. After vortexing to evenly suspend the cells, 100 µL aliquots
were removed from the tubes and spread on LB plates.
9. The plates were incubated at 37˚C for 24 hours.
10. The resulting colonies were counted visually. Each colony
was assumed to have arisen from one cell.
Liquid Exposure Table (Staph)
0%
Vitamin D
0.1 %
Vitamin D
1%
Vitamin D
10%
Vitamin D
154
182
140
106
160
179
137
103
159
172
131
92
174
187
134
97
163
194
144
88
166
176
136
148
184
127
180
Number of Colonies
Liquid Exposure Graph (Staph)
200
180
160
140
120
100
80
60
40
20
0
P-Value = 1.43E-15
Significant
0%
0.10%
1%
Vitamin D Concentration
10%
Dunnett’s Test (Staph)
T-Critical = 2.88
Alpha = 0.05
Concentration of
Vitamin D
T-Value
Significant?
0.1%
5.79
Significant
1%
6.99
Significant
10%
15.50
Significant
Liquid Exposure Table (E. coli)
0%
0.1%
1%
10%
139
189
96
77
148
176
102
82
137
177
113
79
142
168
109
70
153
184
92
86
136
140
106
75
130
180
103
147
169
110
Liquid Exposure Graph (E. coli)
180
P-Value = 1.01E-15
Significant
Number of colonies
160
140
120
100
80
60
40
20
0
0%
0.10%
1%
Concentration of Vitamin D
10%
Dunnett’s Test (E. coli)
T-Critical = 2.88
Alpha = .05
Concentration of
Vitamin D
T-Value
Significant?
0.1%
6.46
Significant
1%
7.65
Significant
10%
12.05
Significant
Comparison of Survivorship
(Staph and E. Coli)
Percentage of Remaining Colonies
140%
120%
100%
80%
E. coli
60%
Staph
40%
20%
0%
0%
0.10%
1%
Concentration of Vitamin D
10%
Conclusion
• The alternate hypothesis can be accepted.
• The null hypothesis can be rejected.
• In small quantities, the vitamin D promoted
bacterial growth.
– For both Staph and E. coli.
• In excess, the bacteria were killed by the
vitamin D.
– For both Staph and E. coli.
Limitations
• The plating was not perfectly synchronized.
• Some cells could have had longer or shorter
exposure times.
• The liquid vitamin D was removed from pill
form, so it possibly contained excess pieces of
the capsule.
Extensions
• More trials to create a better basis for
evaluating the results
• Prolonged exposure test using vitamin D
infused into the agar
• Using an antibiotic resistant strain of bacteria
for the test model
References
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http://ods.od.nih.gov/factsheets/vitamind/
http://www.medicalnewstoday.com/articles/161618.php
http://www.webmd.com/vitamins-supplements/ingredientmono-929VITAMIN%20D.aspx?activeIngredientId=929&activeIngredientName=VITAMIN%20
D
http://www.mayoclinic.com/health/vitamin-d/NS_patient-vitamind
http://www.webmd.com/a-to-z-guides/e-coli-infection-topic-overview
http://www.medicalnewstoday.com/articles/68511.php
http://kidshealth.org/kid/stay_healthy/food/ecoli.html
http://www.about-ecoli.com/
http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/
sepidermidis.html
http://microbewiki.kenyon.edu/index.php/Staphylococcus_epidermidis
http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.10
01133
http://wiki.medpedia.com/Staphylococcus_epidermidis