The Effects of Calcium on the Human Microbial

Flora

Libario ObeidGrade 11

Central Catholic High School

Calcium

Most abundant mineral in the body Many important jobs such as:

Secondary messenger Sensory receptor

99% of calcium stored in bones and teeth for strength;1% in blood, muscle and fluid between cells

Calcium needed for: muscular and blood vessels contraction and

expansion hormonal secretion enzymatic activities neural signal transduction Absorption in small intestine and blood Bone metabolism Homeostasis Channel formation and regulation Tubular reabsorption

Problem

Humans consume many different things, but how can we know to what extent these things affect our physiology?

A viable question which can be asked is: Does excess ingested calcium have adverse effects on symbiotic organisms, in particular, the internal flora?

Liquid Calcium Chloride

CaCl2 is a salt of calcium and chlorine; behaves as typical ionic halide, and is solid at room temperature.

Because of its hygroscopic nature, anhydrous calcium chloride must be kept in tightly-sealed air-tight containers.

Serves as source of calcium ions in a solution and is soluble.

Dangers of Excess Calcium

Excess calcium is absorbed by nails, skin, and even tissues where it is not needed

Premature aging, fatigue, depression and some other conditions can be attributed to excess calcium levels in the body

E. coli

Escherichia coli is one of the most common forms of bacteria found in many environments including the intestinal tracts of many mammals.

Utilized as the most studied prokaryote in biological research.

Many of different strains of E.coli, most of which are non-pathogenic. However, there are strains which can produce fatal disease in humans and other mammals.

Staphylococcus epidermidis

Bacteria that is mostly harmless and resides normally on skin and mucous membranes of humans/organisms

Found worldwide, they are a small component of soil microbial flora.

Very important model organism used in modern biological research, as it is one of the most extensively sequenced bacteria

Past Studies The effects of calcium ions on

the growth of Virulent and Avirulent strains of Pasteurella pestis

Virulent strains of P. pestis required 0.002M to 0.004M calcium ions for growth at 37°C in the basal synthetic medium containing 0.02M magnesium salt.

Avirulent strains grew without added calcium.

Purpose

To assess the effects of different percent concentrations of calcium on the survivorship of E. coli and Staphylococcus epidermidis.

Hypothesis

Null: Survivorship of E. coli and S. epidermidis in varying concentrations of Calcium Chloride will not vary significantly from the control.

Alternative: Survivorship of E. coli and S. epidermidis in varying concentrations of Calcium Chloride will vary significantly from the control.

Materials

40 LB agar plates (1% trytone, 5% yeast extract, 1% NaCl, 2mL 1M NaOH, 1.5% agar)

LB media (1% tryptone, 5% yeast extract, 1% NaCl) Klett spectrophotometer Sterile pipette tips Micropipettes Vortex Incubator Sidearm flask Spreading platform, spreader bar, ethanol 20 mL Sterile capped test tubes with Sterile Dilution Fluid (SDF)

(10mM KH2PO4, 10 mM K2HPO4, 1mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)

Escherichia coli Staphylococcus epidermidis Calcium Chloride [10%] 0.22 micron syringe filters + 10 mL syringe

Procedure

1. Bacteria 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 a shaking water bath until a

density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108-9 cells/mL.

4. The cell culture was diluted in sterile dilution fluid to a concentration of approximately 103 cells/mL.

5. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL.

6. The tubes were prepared as follows:

Concentration 0% 0.01% 0.10% 1%Sterile Dilution

Fluid (SDF) 9.9 mL 9.89 9.8 8.9

E. coli/Staph 0.1 0.1 0.1 0.1Calcium Chloride

[10%] 0 0.01 0.1 1

Total 10 10 10 10

Test Tube Ingredients

Procedure Cont.

6. 0.1 mL of cell culture was then added to the test tubes, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.

7. The solution was mixed by vortexing and was allowed to sit at room temperature for 15 minutes.

8. After vortexing to evenly suspend cells, 0.1 mL 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. Each colony is

assumed to have arisen from one cell.

P value=0.004284748

Dunnett’s Test

S. epidermidis

 Percent Calcium

t-value Interpretation

0.01% 0.6726Not

Significant

0.1%3.154

Significant

1%4.051

Significant

T Crit.= 2.68

E. coli

 Percent Calcium

t-value Interpretation

0.01% 0.7605Not

Significant

0.1%1.942 Not

Significant

1%3.699

Significant

Interpretation

The statistical analyses suggested a correlation between the percent concentration of calcium and cell survivorship. Higher concentrations of calcium chloride resulted in more surviving colonies.

Null hypothesis was rejected for all variables because the p-values were less than the Alpha (0.05)

Conclusion The calcium chloride has an effect

on E. coli and S. epidermidis survivorship.

The survivorship of the variables significantly varied from the control and so the alternative hypothesis was accepted.

The 0.1% and 1% solutions had a significant positive effect on S. epidermidis survivorship.

The 1% solutions had a significant positive effect on E. coli.

Limitations

Only 4 different concentrations were used.

This experiment was limited in that pure calcium was not able to be used.

Plating was not exactly synchronized, which could have resulted in extra time for bacterial replication.

Extensions

Varying exposure time of E. coli and S. epidermidis in the tubes before plating could be tested.

To allow for longer exposure, calcium could be infused directly into the LB agar.

Using different concentrations of calcium.

Using various prokaryotic models

References

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC290369/pdf/jbacter00500-0085.pdf

http://www.about-ecoli.com/ http://web.uconn.edu/mcbstaff/graf/Student

%20presentations/S%20epidermidis/sepidermidis.html http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/

flora/cases.html http://www.oxy.com/our_businesses/chemicals/pages/

chem_products_basic_calciumchloride.aspx http://www.natural-health-information-centre.com/

calcium.html http://www.buzzle.com/articles/too-much-calcium-side-

effects.html