1. Vitamin D Effects on Microbial Flora
Justin Beiriger
Central Catholic High School
Grade 10
2nd Year in PJAS

2. 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.

3. 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.

4. Vitamin D Toxicity 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

5. 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.

6. Previous Studies Edward Mellanby first discovered vitamin D.
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.

7. 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

8. Staphylococcous epidermidis
Human skin flora.
Gram (+) bacteria.
Surrounded by a simple cell wall.
Most forms are non-pathogenic.
Forms biofilms on plastic devices.

9. Purpose
To determine what effect vitamin D has on E. coli and Staph survivorship.

10. 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.

11. Materials
LB agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride)
Bunsen burner
Vortex
Vitamin D (liquid supplement)
Escherichia coli (DH5-alpha)
Staphylococcous epidermidis
Micropipettes
Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
Klett Spectrophotometer
Turntable
Labeling tape
Micro rack
Sterile test tubes
Micro tubes
Sterile spreader bars
0.22 micron sterile filter
Incubator
Ethanol

12. Procedure
Bacteria (E. coli and Staph) was grown overnight in sterile LB media.
A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
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.
The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
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%.

13. Table of Concentrations0%
Vitamin D
0.1% Vitamin D 1%
10% Vitamin D
Microbe
0.1 mL
SDF
9.9 mL
9.89 mL
9.8 mL
8.9 mL
0 mL
0.01 mL
1 mL
Total
10 mL

14. Procedure
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.
The solutions were vortexed and allowed to sit at room temperature for a 20 minute incubation period.
After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB plates.
The plates were incubated at 37˚C for 24 hours.
The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

15. Liquid Exposure Table (Staph)
0% Vitamin D
0.1 %
Vitamin D 1%
10%
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

16. Liquid Exposure Graph (Staph)
P-Value = 1.43E-15
Significant

17. 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
10%
15.50

18. 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

19. Liquid Exposure Graph (E. coli)
P-Value = 1.01E-15
Significant

20. 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
10%
12.05

21. Comparison of Survivorship (Staph and E. Coli)

22. 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.

23. 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.

24. 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

25. References http://ods.od.nih.gov/factsheets/vitamind/