1. The Effects of Calcium on the Human Microbial Flora Libario Obeid Grade 11 Central Catholic High School

2. 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

3. 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?

4. Liquid Calcium Chloride CaCl 2 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.

5. 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

6. 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.

7. 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

8. 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.

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

10. 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.

11. 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 KH 2 PO 4, 10 mM K 2 HPO 4, 1mM MgSO 4, 0.1 mM CaCl 2, 100 mM NaCl) Escherichia coli Staphylococcus epidermidis Calcium Chloride [10%] 0.22 micron syringe filters + 10 mL syringe

12. 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 10 8-9 cells/mL. 4. The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 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:

13. Concentration0%0.01%0.10%1% Sterile Dilution Fluid (SDF)9.9 mL9.899.88.9 E. coli/Staph0.1 Calcium Chloride [10%]00.010.11 Total10 Test Tube Ingredients

14. 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 10 3 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.

15. P value= 0.004284748

17. Dunnett’s Test S. epidermidis Percent Calcium t-valueInterpretation 0.01%0.6726 Not Significant 0.1% 3.154 Significant 1% 4.051 Significant T Crit.= 2.68 E. coli Percent Calcium t-valueInterpretation 0.01%0.7605 Not Significant 0.1% 1.942Not Significant 1% 3.699 Significant

18. 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)

19. 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.

20. 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.

21. 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

22. References http://www.ncbi.nlm.nih.gov/pmc/articles/PMC290369/pdf/jbact er00500-0085.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/ca ses.html http://www.oxy.com/our_businesses/chemicals/pages/chem_pr oducts_basic_calciumchloride.aspx http://www.natural-health-information-centre.com/calcium.html http://www.buzzle.com/articles/too-much-calcium-side- effects.html