1. Antimicrobial Effects of Colloidal Copper Teddy Larkin 11 th Grade Pittsburgh Central Catholic High School

2. 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.

3. 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. 3

4. – 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. Mesocopper

5. 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.

6. 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. 6

7. 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. 7

8. 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. 8 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. Alternate Hypotheses Null Hypotheses

9. Materials 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 9

10. Procedure 1: Pulse Liquid Exposure 1. E. coli DH5-Alpha and Staphylococcus Epidermidis were grown overnight in separate sterilized media. 2. Samples of the overnight cultures were added to fresh LB media in sterile sidearm flasks. 3. The cultures were incubated at 37°C until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells per mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 5. A colloidal copper suspension was mixed with the appropriate amount of SDF to create concentrations of 0%, 0.5%, 1%, 2%, and 5%.

11. Procedure 1 Cont… 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL and placed in test tubes. 5. The colloidal copper was diluted in SDF to create a concentration of 1% solution 6. 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 10 3 cells/mL. 7. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB Agar plates. 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.

12. Chart of Concentration 0%0.5%1.0%2.0%5% Microbe 0.1 ml SDF 9.9 ml9.75 ml9.6 ml9.3 ml8.4 ml Colloidal Copper 0 ml.15 ml0.3 ml0.6 ml1.5ml Total 10 ml 12

13. P Value: 4.11732E-15 P Value: 9.51692E-24

14. 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.

15. ANOVA & Dunnett’s Test Results ANOVA0.5% Copper Solution 1% Copper Solution 2% Copper Solution 5% Copper Solution Staph 4.11732E-15 p<.05 T Values 3.867.1211.418.6 E. Coli 9.51692E-24 p<.05 T Values23.930.637.241.2 T Critical Values: p<0.05:2.83

16. 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 10 8 cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL and placed in test tubes.

17. 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. Procedure 2 Cont…

19. 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. 19

20. 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. 20

21. Sources Bukhari, Mohammad. "Student Presentation on Staphylococcus Epidermis." Web. 2 Nov. 2009.. "Colloidal Copper General Information." Web. 25 Oct. 2009.. "Colloidal Copper Studies-University of North Texas and SilverKare." Web. 28 Oct. 2009.. "Colloidal Copper kills over disease causing bacteria." Kombacha Power Products. Web. 28 Oct. 2009.. "E. coli." Kids Health from Nemours. Web. 28 Oct. 2009.. 21