Transcript Larkin Finalized_Antimicrobial_Effects_of_Copper_Teddy_Larkin

Antimicrobial Effects of Colloidal Copper
Teddy Larkin
11th Grade
Pittsburgh Central Catholic High School
Copper Antimicrobial Effects
• Copper is antibacterial through the oligodynamic
effect.
• The exact mechanism of this effect is still unknown
therefore its definition is unknown.
• Data from silver suggest that these ions denature
enzymes of the target cell or organism by binding to
reactive groups, resulting in their precipitation and
inactivation
• This antimicrobial effect is shown by ions of: mercury,
silver, copper, iron, lead, zinc, gold, aluminum and other
metals.
Copper Uses
• Supports healthy cartilage and tendon regeneration.
• Plays a critical role in cellular energy production.
• Helps maintain the integrity of connective tissue in the heart and
blood vessels.
• Plays an important role in bone formation.
• Plays an important role in the metabolism of some major
neurotransmitters.
• Functions as an antioxidant.
• Necessary for normal iron metabolism and red blood cell formation.
• Is known to play an important role in the development and
maintenance of immune system function.
• Increases the body's ability to absorb iron.
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Mesocopper
–All natural mineral supplement
–In the form of a copper colloid
consisting of nanometer particles of
0.9999 pure copper suspended in pure
deionized water.
–Contains 10 parts-per-million (PPM) of
copper nanoparticles.
Escherichia Coli
• Rod-shaped, 2 micrometers
diameter
• Gram negative bacteria
• reproduction rate: 20 min
• Survival, growth, and replication
require only a single carbon source
and ammonium salts.
• E. Coli: pathogen that is found in
the lower intestines of warm
blooded animals.
• Almost 73,000 cases of infection
and 61 deaths per year in the
United States.
• Most common prokaryote model.
Staphylococcus Epidermidis
• Gram positive coccus.
• Common surface symbiont in many mammals
(including humans).
• Most forms considered non-pathogenic.
• Potentially pathogenic upon systemic entry.
• Forms biofilms.
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Purpose
To assess the effects of Colloidal
Copper exposure on the
survivorship of E. Coli and Staph
Epidermidis through
implementation of agar infusion
and pulse liquid exposure.
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Null Hypotheses
1. Prolonged colloidal copper exposure (agar infusion)
will not significantly reduce the survivorship of E.
coli and Staph Epidermidis.
2. Colloidal copper pulse liquid exposure will not
significantly reduce the survivorship of E. coli and
Staph Epidermidis.
Alternate Hypotheses
1. Prolonged colloidal copper exposure through agar
infusion will significantly reduce the survivorship of E.
Coli or Staph Epidermidis.
2. Colloidal copper pulse liquid exposure will significantly
reduce the survivorship of E. coli and Staph
Epidermidis.
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Materials
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Mesocopper
Beaker
Micro and macro pipettes + tips
Spreader bars
96 LB agar plates (1% Tryptone, .5% Yeast Extract, 1% NaCl)
Escherichia Coli bacteria
Staphylococcus Epidermis bacteria
Burner
Turn-table
Tube racks
Vortex
Incubator
Gloves\goggles
SDF (sterile dilution fluid)
Sterile 9ml test tubes
Ethanol
Klett spectrophotometer
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Procedure 1: Pulse Liquid Exposure
1. E. coli DH5-Alpha and Staphylococcus Epidermidis
2.
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4.
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were grown overnight in separate sterilized media.
Samples of the overnight cultures were added to
fresh LB media in sterile sidearm flasks.
The cultures were incubated at 37°C until a
density of 50 Klett spectrophotometer units was
reached. This represents a cell density of
approximately 108 cells per mL.
The culture was diluted in sterile dilution fluid to a
concentration of approximately 105 cells/mL.
A colloidal copper suspension was mixed with the
appropriate amount of SDF to create
concentrations of 0%, 0.5%, 1%, 2%, and 5%.
Procedure 1 Cont…
4. The culture was diluted in sterile dilution fluid to a
5.
6.
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concentration of approximately 105 cells/mL and placed in
test tubes.
The colloidal copper was diluted in SDF to create a
concentration of 1% solution
100 µL of cell culture was then added to the copper
solutions, yielding a final volume of 10 mL and a cell density
of approximately 103 cells/mL.
After vortexing to evenly suspend the cells, 100 µL aliquots
were removed from the tubes and spread on LB Agar 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.
Chart of Concentration
Microbe
SDF
0%
0.5%
1.0%
2.0%
5%
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
9.9 ml 9.75 ml
9.6 ml
9.3 ml
8.4 ml
Colloidal
Copper
0 ml
.15 ml
0.3 ml
0.6 ml
1.5ml
Total
10 ml
10 ml
10 ml
10 ml
10 ml
12
Colonies
Pulse Exposure - Bacterial Survivorship
800
700
600
500
400
300
200
100
0
P Value: 4.11732E-15
P Value: 9.51692E-24
E. coli
Staph
0%
0.50%
1%
2%
Concentration of colloidal copper
5%
ANOVA & Dunnett’s Tests
• An ANOVA (analysis of variance) is a statistical analysis which
allows you to measure the means of groups of data.
• If the ANOVA has a significant p value a Dunnett’s test can be
performed.
• The MSE (Mean Square Error) value from the ANOVA then is
used in the Dunnett’s test.
• Allows comparison of each experimental group to the control.
• Determines if there is a significant difference between the
control and the experimental groups.
ANOVA & Dunnett’s Test Results
T Critical Values: p<0.05:2.83
ANOVA
Staph
0.5% Copper
Solution
1% Copper
Solution
2% Copper
Solution
5% Copper
Solution
p<.05
p<.05
p<.05
p<.05
T Values
3.86
7.12
11.4
18.6
E. Coli
p<.05
p<.05
p<.05
p<.05
23.9
30.6
37.2
41.2
4.11732E-15
9.51692E-24
T Values
Prolonged Exposure (agar infusion)
Procedure 2
1. 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 and placed in test tubes.
Procedure 2 Cont…
5. The colloidal copper was diluted in SDF to create a
concentration of 1% solution
6. Then 200 uL of the 1% copper solution was infused
into all 6 infused plates.
7. After infusion, 100 µL aliquots were taken from the
bacteria tubes and spread on 6 LB Agar plates and 12
infused plates, 6 plates per bacteria.
8. The plates were incubated at 37°C for 24 hours.
9. The resulting colonies were counted visually. Each
colony was assumed to have arisen from one cell.
Agar InfusionBacteria Survivorship
Dunnett’s Test- Infusion
200
ANOVA
180
1% Copper
Solution
160
Staph
Colonies
140
1.61931E-05
p<.01
120
T Values
100
7.71
Staph
80
E. coli E. Coli
2.88163E-06
p<.01
60
40
T Values
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14.6
0
0%
1%
Colloidal Copper
Concentration
T Critical Values: p<0.05: 2.34
p<.01: 3.61
Conclusions
1. The null hypothesis was rejected in prolonged
colloidal copper exposure (agar infusion) since
the variable significantly reduced the
survivorship of E. coli and Staphylococcus
Epidermidis.
2. The null hypothesis was rejected at all values in
colloidal copper pulse liquid exposure since the
variable significantly reduced the survivorship of
E. coli and Staphylococcus Epidermidis.
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Extensions
Limitations
Test a higher
concentration of
colloidal copper
on bacteria.
Test even broader
types of bacteria.
Only one
concentration of
infused colloidal
copper was
tested.
There was a lag
time when plating
the cells.
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Sources
 Bukhari, Mohammad. "Student Presentation on Staphylococcus Epidermis." Web. 2
Nov. 2009.
<http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis
/sepidermidis.html>.
 "Colloidal Copper General Information." Web. 25 Oct. 2009.
<http://www.lifedevice.com/General%20Info.htm>.
 "Colloidal Copper Studies-University of North Texas and SilverKare." Web. 28 Oct.
2009. <http://www.silvermedicine.org/colloidalsilverstudytexas.html>.
 "Colloidal Copper kills over disease causing bacteria." Kombacha Power Products.
Web. 28 Oct. 2009. <http://www.kombuchapower.com/colloidal_copper.htm>.
 "E. coli." Kids Health from Nemours. Web. 28 Oct. 2009.
<http://kidshealth.org/kid/stay_healthy/food/ecoli.html#>.
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