Vitamin D Effects on Microbial Flora

Justin BeirigerCentral Catholic High SchoolGrade 102nd 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

• 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

Procedure1. 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%.

0% Vitamin D

0.1% Vitamin D

1% Vitamin D

10% 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

Table of Concentrations

Procedure6. 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 D0.1 %

Vitamin D1%

Vitamin D10%

Vitamin D154 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

Liquid Exposure Graph (Staph)

0.00% 0.10% 1.00% 10.00%0

20406080

100120140160180200

Vitamin D Concentration

Num

ber o

f Col

onie

s

P-Value = 1.43E-15Significant

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)

0.00% 0.10% 1.00% 10.00%0

20

40

60

80

100

120

140

160

180

Concentration of Vitamin D

Num

ber o

f col

onie

s

P-Value = 1.01E-15Significant

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)

0.00% 0.10% 1.00% 10.00%0%

20%

40%

60%

80%

100%

120%

140%

E. coliStaph

Concentration of Vitamin D

Perc

enta

ge o

f Rem

aini

ng C

olon

ies

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• http://ods.od.nih.gov/factsheets/vitamind/• http://www.medicalnewstoday.com/articles/161618.php• http://www.webmd.com/vitamins-supplements/ingredientmono-929-VITAMIN

%20D.aspx?activeIngredientId=929&activeIngredientName=VITAMIN%20D• 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.1001133• http://wiki.medpedia.com/Staphylococcus_epidermidis