1. Alex Senchak Grade 9 Central Catholic High School 1 Colloidal Silver Antibacterial Assessment

2.  Bacteria that cause infections are becoming increasingly resistant to antibiotics and other antimicrobial agents. 2

3.  One of the most common forms of bacteria found in many environments.  Symbiont in intestinal tracts of many mammals.  Gram negative, rod shaped bacillus.  Most non-pathogenic.  Pathogenic strains can lead to life threatening infections. 3

4.  Gram positive coccus.  Common surface symbiont in many mammals (human).  Most forms considered non- pathogenic.  Pathogenic forms can be life threatening.  Forms biofilms. 4

5. Gram Positive (Staph)Gram Negative (E. Coli)  Cell wall is thin extra layer of lipopolysaccharide which adds extra level of protection.  If the toxin enters the circulatory system it causes a toxic reaction.  This outer membrane protects the bacteria from several antibiotics. 5  Most pathogenic bacteria in humans are gram-positive organisms.  Simple cell wall.  Antibiotics such as penicillin work against the formation of the cell wall.

6.  Disables enzymes that power bacteria.  Colloidal Silver as an antibiotic.  Argyria: Caused by overuse of Colloidal Silver. 6 Paul Karason “Blue Man”

7.  To determine if Colloidal Silver will affect the survivorship of E. Coli and Staphylococcus Epidermidis. 7

8. Colloidal Silver exposure will not significantly reduce the survivorship of E. Coli or Staphylococcus Epidermidis. 8 Hypothesis Colloidal Silver exposure will significantly reduce the survivorship of E. Coli and Staphylococcus Epidermidis.

9. Large glass jar Beaker Micro and macro pipettes + tips Spreader bars 76 LB agar plates (1% Tryptone, 0.5% Yeast Extract, 1% NaCl) Escherichia Coli bacteria Staphylococcus Epidermidis bacteria Colloidal Silver making kit: Generator Silver Electrodes Buffer TDS meter Burner Turn-table Tube racks Vortex Incubator Gloves\goggles Klett Spectrophotometer SDF (sterile dilution fluid). 20 sterile test tubes Ethanol 9

10. 1. Sterile glass container, beaker and measuring cup were used to transfer liquid. 2. Used measuring cup to pour approximately 470 ml of distilled water into the glass container. 3. Attached silver electrodes to generator. 4. Electrodes were suspended in the solution and powered for 75 minutes. 5. This process was performed in the dark for efficiency. 6. The deposition of dark particles on the electrodes provided evidence of colloidal silver. 7. After 75 minutes a TDS meter was used to determine the concentration of silver in the solution. 8. The silver solution was stored in the dark until use. 10

11. 11

12. 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. 5. A colloidal silver suspension was mixed with the appropriate amount of SDF to create colloidal silver concentrations of 13%, 6.5%, 1.3%, and 0.13%. 12

13. 0% Stock 0.13% Stock 1.3% Stock 6.5% Stock 12.87% Stock Microbe 0.1 ml SDF 9.9 ml 9.8 ml8.9 ml4.9 ml0 ml Colloidal Silver 0 ml0.1 ml1 ml5 ml9.9 ml Total 10 ml 13

14. 6. 100 µL of cell culture was then added to the silver solutions, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 8. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB agar plates. 9. The plates were incubated at 37°C for 24 hours. 10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell. 14

15. 15 Colonies P= 7.53E-24 0% 1.3%6.5%

16. Dunnett’s Test Analysis (E. coli) T Critical = 3.26 (significant) Alpha =.05 Silver Concentration T ValueInterpretation 0.13% 6.75 Significant 1.3% 12.92 Significant 6.5% 15.91 Significant 12.87% 42.82 Significant 16

17. 17 Colonies P= 3.75E-19 0%

18. Dunnett’s Test Analysis (Staph) T Critical = 3.26 (significant) Alpha =.05 Silver Concentration T ValueInterpretation 0.13% 13.69 Significant 1.3% 18.41 Significant 6.5% 22.31 Significant 12.87% 27.85 Significant 18

19. 19 Percentage of Surviving Colonies % of Silver in Solution

20.  There appeared to be a dosage effect of the silver on the bacteria. Increasing amounts of silver led to less bacteria surviving. 20

21. 21Conclusion Null Hypothesis Colloidal Silver exposure will not significantly reduce the survivorship of E. Coli or Staphylococcus Epidermidis. REJECTED Hypothesis Colloidal Silver exposure will significantly reduce the survivorship of E. Coli and Staphylococcus Epidermidis. ACCEPTED

22.  Test a higher concentration of colloidal silver on bacteria.  Test different types of bacteria.  Infuse the agar with silver to provide a prolonged surface exposure.  There was a lag time when plating the cells. Exposure times to the silver varied slightly. 22

23. Bukhari, Mohammad. "Student Presentation on Staphylococcus Epidermis." Web. 2 Nov. 2009.. "Colloidal Silver General Information." Web. 25 Oct. 2009.. "Colloidal Silver Studies-Univiversity of North Texas and SilverKare." Web. 28 Oct. 2009.. "Colloidal Slilver kills over disease causing bacteria." Kombacha Power Products. Web. 28 Oct. 2009.. "E. coli." Kids Health from Nemours. Web. 28 Oct. 2009.. "Silver Poisoning & Argyria: FDA, EPA, WHO." Colloidal Silver Safety and Toxicity Center. Web. 3 Nov. 2009.. 23

24. 24 Staph Results

25. 10 2 cells 10 3 cells/mL 10 5 cells/mL with silver 10 8 cells/mL (Bacteria) 100 uL 10 7 cells/mL (Bacteria) 200 uL400 uL 25