Effect of TAML® Activated
Peroxide on Viral Inhibition
Philip Dulac
Pittsburgh Central
Catholic High School
Sustainability
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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
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Among the largest
sources of industrial
pollution
Ubiquitous
Depends on the use of
heavy metals and
chlorine
Green Chemistry
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An alternative to
oxidation chemistry
Seeks to accomplish
the tasks of oxidation
chemistry without the
hazardous effects
Involves use of nontoxic substances often
made of the elements
of life
TAML® Activators
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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
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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
Plaques
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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
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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
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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
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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.
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
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.
Procedure with H2O2
12. The microtubes were made using the following grid (the T2 phage
concentration was reduced to ~102 phages/mL).
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
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.
Procedure with H2O2, continued
15. The experimental microtubes were made using the following grid (the
T2 phage concentration was reduced to ~102 phages/mL).
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
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.
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®
80
Average Number of Plaques
70
Water Only
60
1mM H2O2
50
40
1mM H2O2 +
catalase
30
100mM
H2O2
20
100mM
H2O2 +
catalase
10
0
1 minute
5 minutes
15 minutes
Time
30 minutes
5-minute Sets Without H2O2
70
62
Average Number of Plaques
60
58
60
57
50
40
5 minutes
30
20
10
0
Water only
50uM TAML
catalase
Set
50uM TAML +
catalase
TAML® and H2O2 in Combination
50
Average Number of Plaques
45
40
35
1mM H2O2 +
1uM TAML
1mM H202 +
50uM TAML
100mM H2O2
+ 1 uM TAML
100mM H2O2
+ 50uM TAML
30
25
20
15
10
5
0
1 minute
5 minutes
15 minutes
Time
30 minutes
ANOVA Statistical Analysis
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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 5minute 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
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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
150
Average Number of Plaques
135
120
105
90
100mM H2O2
+ 1 uM TAML
75
100mM H2O2
+ 50 uM TAML
60
45
30
15
0
15 minutes
30 minutes
Time
45 minutes
Conclusions
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Increasing concentrations of TAML® and hydrogen
peroxide decreased the infectivity of T2 phages.
This conclusion was further supported by the low pvalues 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
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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
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/ 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.Coli
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