Transcript Obeid The Effects of Calcium on the Human Microbial

The Effects of Calcium on
the Human Microbial Flora
Libario Obeid
Grade 11
Central Catholic High School
Calcium
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Most abundant mineral in the body
Many important jobs such as:
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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:
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muscular and blood vessels contraction and expansion
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hormonal secretion
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enzymatic activities
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neural signal transduction
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Absorption in small intestine and blood
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Bone metabolism
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Homeostasis
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Channel formation and regulation
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Tubular reabsorption
Problem
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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
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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 tightlysealed air-tight containers.
Serves as source of calcium ions
in a solution and is soluble.
Dangers of Excess Calcium
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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
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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
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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
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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
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To assess the effects of
different percent
concentrations of calcium
on the survivorship of E.
coli and Staphylococcus
epidermidis.
Hypothesis
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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
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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
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Bacteria 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 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.
The cell culture was diluted in sterile dilution fluid to a
concentration of approximately 103 cells/mL.
The selected experimental variables were diluted with sterile
dilution fluid to the chosen concentrations to a total of 9.9 mL.
The tubes were prepared as follows:
Test Tube Ingredients
Concentration
0%
0.01%
Sterile Dilution
Fluid (SDF)
9.9 mL 9.89
0.10%
1%
9.8
8.9
E. coli/Staph
Calcium Chloride
[10%]
0.1
0.1
0.1
0.1
0
0.01
0.1
1
Total
10
10
10
10
Procedure Cont.
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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.
The solution was mixed by vortexing and was allowed to sit at
room temperature for 15 minutes.
After vortexing to evenly suspend cells, 0.1 mL 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. Each colony is assumed
to have arisen from one cell.
P value=
0.004284748
Dunnett’s Test
T Crit.= 2.68
S. epidermidis
Percent
Calcium
t-value
Interpretation
0.01%
0.6726
0.1%
1%
3.154
4.051
E. coli
Percent
Calcium
t-value
Interpretation
Not
Significant
0.01%
0.7605
Not
Significant
Significant
0.1%
Significant
1%
1.942
3.699
Not
Significant
Significant
Interpretation
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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
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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
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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
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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
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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-sideeffects.html