Effect of TAML® Activated Peroxide on Viral Inhibition

Philip Dulac

Pittsburgh Central Catholic High School

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

Oxidation Chemistry

Among the largest sources of industrial pollution

Ubiquitous Depends on the use of

heavy metals and chlorine

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

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

The TAML® Catalyst

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% DNAT2 Phage

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

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.

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.

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 1.8x108 phages/ml)

Sterile microtubes Sterile water 250-mL sterile sidearm flask

Sterile DCBF2 TAML® solution (initial concentration 5x10-4M)

Sterile syringe filters Sterile 8.8M H2O2 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

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 103 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.

Procedure without H2O2

9. The volume in each of the following microtubes was 1.0 mL, reducing the T2 phage concentration to ~102 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®] [H2O2] Total Plates(triplicates)

Catalase

1 0M 0M 12 0μL

2 50μM 0M 3 0μL

3 50μM 0M 3 10μL

4 0M 0M 3 10μL

Procedure with H2O2

12. The microtubes were made using the following grid (the T2 phage concentration was reduced to ~102 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®] [H2O2] Total Plates(triplicates)

Catalase

1 0M 1 mM 12 0μL

2 0M 100 mM 12 0μL

3 0M 1 mM 12 10μL

4 0M 100 mM 12 10μL

Procedure with H2O2, continued

15. The experimental microtubes were made using the following grid (the T2 phage concentration was reduced to ~102 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®] [H2O2] Total Plates(triplicates)

Catalase

1 1μM 1 mM 12 10μL

2 50μM 1 mM 12 10μL

3 1μM 100 mM 12 10μL

4 50μM 100 mM 12 10μL

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.

Sets without TAML®

0

10

20

30

40

50

60

70

80

1 minute 5 minutes 15 minutes 30 minutes

Time

Avera

ge N

um

ber

of

Pla

qu

es

Water Only

1mM H2O2

1mM H2O2 +catalase

100mMH2O2

100mMH2O2 +catalase

5-minute Sets Without H2O2

6258 57

60

0

10

20

30

40

50

60

70

Water only 50uM TAML catalase 50uM TAML +catalase

Set

Avera

ge N

um

ber

of

Pla

qu

es

5 minutes

TAML® and H2O2 in Combination

0

5

10

15

20

25

30

35

40

45

50

1 minute 5 minutes 15 minutes 30 minutes

Time

Avera

ge N

um

ber

of

Pla

qu

es

1mM H2O2 +1uM TAML1mM H202 +50uM TAML

100mM H2O2+ 1 uM TAML100mM H2O2+ 50uM TAML

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%.

Results of Some ANOVA Analyses

Analysis P value Accept or Reject Null? Explanation

Water – all time intervals 0.570405 Accept This analysis showed that water did not affect viral survivorship over time.

Water (5 minutes) vs. other 5-minute sets without H2O2

0.518519 Accept This analysis showed that 50μM TAML®, catalase, and 50μM TAML® + catalase cannot

work without H2O2.

Water vs. [1mM H2O2 + 50μM

TAML®] (all time intervals)6.46E-10 Reject This analysis showed that this concentration of

TAML® and hydrogen peroxide has greatly affected viral infectivity.

Water vs. [100mM H2O2 +

1μM TAML®] (all time intervals)

8.93E-13 Reject This analysis showed that this concentration of TAML® and hydrogen peroxide has greatly

affected viral infectivity.

Water vs. [100mM H2O2 +

50μM TAML®] (all time intervals)

1.93E-13 Reject This analysis showed that this concentration of TAML® and hydrogen peroxide has greatly

affected viral infectivity.

[100mM H2O2 + catalase] vs.

[100mM H2O2 + 1μM

TAML®] (all time intervals)

8.78E-12 Reject This data analysis shows that in the same concentration of H2O2, the addition TAML®

greatly decreases viral infectivity.

[100mM H2O2 + catalase] vs.

[100mM H2O2 + 50μM

TAML®] (all time intervals)

1.05E-12 Reject This data analysis shows that in the same concentration of H2O2, the addition TAML®

greatly decreases viral infectivity.

Higher Phage Concentrations

Because of the success of the TAML® catalyst in combination with 100mM H2O2, a trial was run with a higher concentration of T2 phages.

The corresponding procedures were repeated, except a T2 phage stock of 107 phages/mL was used.

Inactivation of 106 phages/mL

0

15

30

45

60

75

90

105

120

135

150

15 minutes 30 minutes 45 minutes

Time

Avera

ge N

um

ber

of

Pla

qu

es

100mM H2O2+ 1 uM TAML

100mM H2O2+ 50 uM TAML

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.

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

Bibliography

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2. Collins, Terry. “Institute for Green Oxidation Chemistry.” © 2001 Carnegie-Mellon Universityhttp://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.Coli5. “Green Chemistry.” © 2004 Interuniversity Consortium

http://venus.unive.it/inca/research/green_chemistry/index.php6. “Oxidation Chemistry and Redox Reactions.” ©2006.

Wikipedia Online Encyclopedia. http://en.wikipedia.org/wiki/redox7. Safarzadeh-Amiri, A., J. R. Bolton, and S. R. Cater. The Use of Iron in

Advanced Oxidation Processes. © 1996. Journal of AdvancedOxidation. 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 coliIn Photocatalytic Disinfection. ©Jan 2005. Journal of Applied and Environmental Microbiology