1. Effect of TAML ® Activated Peroxide on Viral Inhibition Philip Dulac Pittsburgh Central Catholic High School

2. Sustainability An issue facing our world today Ultimate goal – meet the needs of the present generation while allowing future generations to successfully meet their own needs

3. Oxidation Chemistry Among the largest sources of industrial pollution Ubiquitous Depends on the use of heavy metals and chlorine

4. Green Chemistry An alternative to oxidation chemistry Seeks to accomplish the tasks of oxidation chemistry without the hazardous effects Involves use of non- toxic substances often made of the elements of life

5. TAML ® Activators Developed by scientists at CMU, led by chemistry professor Dr. Terry Collins Water-soluble, easy to use, and work over a broad pH range Work with hydrogen peroxide Biodegradable and engineered to self-destruct after performing their function Capable of destroying biological warfare agents at low concentrations

6. The TAML ® Catalyst

7. T2 Phage Used as a model to examine if TAML ® can disinfect water and dry surfaces Historically, the T-even series of phages have been used by scientists because they are safe and relatively easy to quantify. 50% protein and 50% DNA T2 Phage

8. Plaques Created when a T2 phage attacks its E. Coli host Inverses of colonies – no E. Coli growth (in a radial distance from the first active phage) Indicate an active virus that infected its host and reproduced through the lytic cycle E. Coli Plaques

9. Purpose This experiment was designed to determine if TAML ® Activators can deactivate viruses when in solution with hydrogen peroxide. If that is the case, it will be investigated whether activated TAML ® is more effective than hydrogen peroxide at lowering viral infectivity.

10. Hypotheses Increasing concentrations of TAML ® and hydrogen peroxide will result in lower T2 phage survivorship. As exposure time increases, TAML ® and hydrogen peroxide will deactivate more T2 phages. The null hypothesis states that no variation in infectivity will be present.

11. Materials List Autoclave Sterile LB Media (E. coli) – Contains 1% tryptone,.5% yeast extract, 1% NaCl, 2ml of 1M NaOH (per liter) 15 grams of sterile LB agar (per E. Coli plate) Sterile top agar – Contains 8g Difco Bacto Nutrient Broth, 8g Difco Bacto Nutrient Agar, 5g NaCl (per liter) E. coli B host – in log phase at a cell density of 100-150 Klett spectrophotometer units T2 Phage (purchased from Ward’s Supplyhouse; initial concentration is 1.8x10 8 phages/ml) Sterile microtubes Sterile water 250-mL sterile sidearm flask Sterile DCBF 2 TAML ® solution (initial concentration 5x10-4M) Sterile syringe filters Sterile 8.8M H 2 O 2 purchased from Sigma Aldrich Catalase from Aspergillus niger Sterile pipettes Bunsen burner Incubator Vortex Klett Spectrophotometer 70% Ethanol Thermometer Hot water bath (45°C) Sterile 15-mL polystyrene conicals Microwave Stopwatch Shaker and inoculating loop

12. Procedure: Preparation Work 1. An E. Coli culture was prepared in LB media. 2. The sidearm flask was placed in a shaking water bath. 3. Using a Klett Spectrophotometer, it was ensured that the E. Coli was in log phase (100-150 Klett units). 4. The LB agar plates were pre-heated in an incubator. 5. A solution of 10 3 phages/mL was prepared using sterile water and the T2 phage stock solution. 6. A hot water bath was set to 45°C. 7. The top agar was liquefied in a microwave. 8. 3.0mL of top agar was added to sterile conicles partially submerged in the hot water bath.

13. Procedure without H 2 O 2 9. The volume in each of the following microtubes was 1.0 mL, reducing the T2 phage concentration to ~10 2 phages/mL. 10. For tube 1, a 0.1mL aliquot was extracted at 1, 5, 15, and 30-minute time intervals to make plates using the overlay technique. 11. For tubes 2-4, extractions took place at a 5-minute time interval only. Tube[TAML ® ][H 2 O 2 ]Total Plates (triplicates) Catalase 10M 120μL 250μM0M30μL 350μM0M310μL 40M 310μL

14. Procedure with H 2 O 2 12. The microtubes were made using the following grid (the T2 phage concentration was reduced to ~10 2 phages/mL). 13. For tubes 1 and 2, aliquots of T2 phages were extracted at 1, 5, 15, and 30-minute time intervals. 14. For tubes 3 and 4, catalase was added at 1, 5, 15, and 30-minute time intervals. Once the reaction was fully quenched, the aliquots of T2 phages were extracted. Tube[TAML ® ][H 2 O 2 ]Total Plates (triplicates) Catalase 10M1 mM120μL 20M100 mM120μL 30M1 mM1210μL 40M100 mM1210μL

15. Procedure with H 2 O 2, continued 15. The experimental microtubes were made using the following grid (the T2 phage concentration was reduced to ~10 2 phages/mL). 16. The reactions were quenched at 1, 5, 15, and 30-minute time intervals with 10 microliters of catalase before making plates using the overlay technique. Once all the oxygen bubbles are gone, hydrogen peroxide, and consequently, TAML ®, will cease to affect the T2 phages remaining. Tube[TAML ® ][H 2 O 2 ]Total Plates (triplicates) Catalase 11μM1 mM1210μL 250μM1 mM1210μL 31μM100 mM1210μL 450μM100 mM1210μL

16. Procedure: Overlay Technique 17. The pre-warmed plates were taken out of the incubator. 18. The desired microtube was inverted to get an even mixture of phages. 19. 0.3mL of E. coli host was added directly from the sidearm flask into one conical partially submerged in the hot water bath. 20. Directly after adding the E. coli, a 0.1mL aliquot from the desired sample was added into the conical. 21. The conical was taken out of the hot water bath and wiped dry to prevent contamination from the water bath fluid. 21. After vortexing, the conical’s contents were poured on an LB agar plate, and the plate was swirled. 22. After the top agar congealed, the plate was incubated at 37 °C for 24 hours. This procedure was repeated two more times to create three replicates. 23. This procedure was repeated for each desired sample. 24. Plaques were counted; each plaque was assumed to have arisen from one active T2 phage. Non-circular marks on the top agar were not counted.

17. Sets without TAML ®

18. 5-minute Sets Without H 2 O 2

19. TAML ® and H 2 O 2 in Combination

20. ANOVA Statistical Analysis Compares the variation between groups to variation within groups. A p-value between 0 and 1 gives a confidence level for statistical significance. The cutoff value for this study was 0.05, corresponding to a variance confidence level of at least 95%.

21. Results of Some ANOVA Analyses AnalysisP valueAccept or Reject Null?Explanation Water – all time intervals 0.570405Accept This analysis showed that water did not affect viral survivorship over time. Water (5 minutes) vs. other 5- minute sets without H 2 O 2 0.518519Accept This analysis showed that 50μM TAML ®, catalase, and 50μM TAML ® + catalase cannot work without H 2 O 2. Water vs. [1mM H 2 O 2 + 50μM TAML ® ] (all time intervals) 6.46E-10Reject This analysis showed that this concentration of TAML ® and hydrogen peroxide has greatly affected viral infectivity. Water vs. [100mM H 2 O 2 + 1μM TAML ® ] (all time intervals) 8.93E-13Reject This analysis showed that this concentration of TAML ® and hydrogen peroxide has greatly affected viral infectivity. Water vs. [100mM H 2 O 2 + 50μM TAML ® ] (all time intervals) 1.93E-13Reject This analysis showed that this concentration of TAML® and hydrogen peroxide has greatly affected viral infectivity. [100mM H 2 O 2 + catalase] vs. [100mM H 2 O 2 + 1μM TAML ® ] (all time intervals) 8.78E-12Reject This data analysis shows that in the same concentration of H 2 O 2, the addition TAML ® greatly decreases viral infectivity. [100mM H 2 O 2 + catalase] vs. [100mM H 2 O 2 + 50μM TAML ® ] (all time intervals) 1.05E-12Reject This data analysis shows that in the same concentration of H 2 O 2, the addition TAML ® greatly decreases viral infectivity.

22. Higher Phage Concentrations Because of the success of the TAML ® catalyst in combination with 100mM H 2 O 2, a trial was run with a higher concentration of T2 phages. The corresponding procedures were repeated, except a T2 phage stock of 10 7 phages/mL was used.

23. Inactivation of 10 6 phages/mL

24. Conclusions Increasing concentrations of TAML ® and hydrogen peroxide decreased the infectivity of T2 phages. This conclusion was further supported by the low p- values of the ANOVA analyses. The data also indicated that TAML ® activated by hydrogen peroxide was more effective than hydrogen peroxide alone. The null hypothesis was rejected for the trials with TAML ® and hydrogen peroxide due to ANOVA p-values well below the cutoff margin.

25. Extensions Running the trials in solutions of varying pH Testing whether TAML ® activators can disinfect a dry surface Using other phages or viruses Examining whether the TAML ® catalyst denatures the T2 phage DNA strand

26. Bibliography 1. “Analysis of Variance.” © 2006 StatPoint, Incorporated. http://www.statgraphics.com/analysis_of_variance.htm 2. Collins, Terry. “Institute for Green Oxidation Chemistry.” © 2001 Carnegie-Mellon University http://www.chem.cmu.edu/groups/collins/http://www.chem.cmu.edu/groups/collins/ and all pages on the site 3. Debartolomeis, J., and V. J. Cabelli. Evaluation of an Escherichia coli Host Strain for Enumeration of Bacteriophages. ©1996. Journal of Applied and Environmental Microbiology. 4. “Escherichia coli.” © 2006 Wikipedia Online. Encyclopedia. http://en.wikipedia.org/wiki/E.Colihttp://en.wikipedia.org/wiki/E 5. “Green Chemistry.” © 2004 Interuniversity Consortium http://venus.unive.it/inca/research/green_chemistry/index.php 6. “Oxidation Chemistry and Redox Reactions.” ©2006. Wikipedia Online Encyclopedia. http://en.wikipedia.org/wiki/redox 7. Safarzadeh-Amiri, A., J. R. Bolton, and S. R. Cater. The Use of Iron in Advanced Oxidation Processes. © 1996. Journal of Advanced Oxidation. Technologies. 8. “What is Sustainability?” © 28 February 2005. http://www.environment.sa.gov.au/sustainability/definitions.html 9. Wonyong Choi, Min Cho, Hyenmi Chung, and Jeyong Yoon. Different Inactivation Behaviors of MS-2 Phage and Escherichia coli In Photocatalytic Disinfection. ©Jan 2005. Journal of Applied and Environmental Microbiology