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Interaction of bacterial spores with radicals generated by microwave and low temperature radio frequency discharges

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microwave and low temperature radio frequency discharges
Lin, Szu-Min, D.Sc.
The University of Texas at Arlington, 1986
Copyright © 1986 by Lin, Szu-Min. A ll rights reserved.
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INTERACTION OP BACTERIAL SPORES WITH
RADICALS GENERATED BY MICROWAVE AND
LOW TEMPERATURE RADIO FREQUENCY DISCHARGES
oy
SZU-MIN LIN
Presented to the Faculty of the Graduate School of
The University of Texas at Arlington in Partial Fulfillment
of “the Requirements
fo:r the Degree of
DOCTOR OF SCIENCE
THE UNIVERSITY CP TEXAS AT ARLINGTON
May 1986
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INTERACTION OF BACTERIAL SPORES WITH
RADICALS GENERATED BY MICROWAVE AND
LOW TEMPERATURE RADIO FREQUENCY DISCHARGES
APPROVED:
(SupeWisilng Professor)
CjauJ
d
u
______
i
t
Q
*
J
—
(Dean of the Graduate School)
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,
®Copyright by Szu-Min Lin 1986
All Rights Reserved
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To Mom and Dad
With love
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ACKNOWLEDGEMENT
I wish to extend my most sincere thanks to Dr. Richard
B. Timmons and Dr. Paul T. Jacobs for their guidance and
invaluable suggestions throughout the course of this study.
I also wish to express my appreciation to the other
members of my supervising committee, Dr. Zoltan A. Schelly,
Dr. Andrew L. Ternay, Jr., Dr. Dennis S. Marynick, and Dr.
Krishnan Rajeshwar, for their helpful comments in the
preparation and completion of this dissertation.
I would also like to thank Mr. Ronald F. Berry for his
general support with the microbiological work.
I am grateful to the staff of the Department of
Chemistry and members of Surgikos Incorporated, Arlington,
Texas and to all of those who assisted me in the field.
Special thanks to my family for their understanding
and encouragement.
January 1, 1986
v
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ABSTRACT
INTERACTION OF BACTERIAL SPORES WITH
RADICALS GENERATED BY MICROWAVE AND
LOW TEMPERATURE RADIO FREQUENCY' DISCHARGES
Publication No. _______
Szu-Min Lin, D.Sc.
The University of Texas at Arlington, 1986
Supervising Professor: Richard B. Timmons
Microwave and radio frequency (RF) discharges were used
to generate active radical species, which were tested for
sporicidal activity.
The microwave discharge studies include
a comparison of the relative sporicidal activity of oxygen,
hydrogen, hydroxyl and hydroperoxyl radicals.
In addition,
the effects of temperature, distance, substrate and packaging
on the sporicidal activity of these species were determined.
The sporicidal activity of a number of chemicals in an
RF discharge was evaluated. Only hydrogen peroxide vapor and
plasma showed an apparent synergism for sporicidal activity.
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The major sporicidal activity of hydrogen peroxide/plasma
appears to he due to the radicals generated during the
process and not the effect of heat on hydrogen peroxide.
A hydrogen peroxide pretreatment period prior to the use
of plasma was found to enhance sporicidal activity.
Pre­
sumably, this pretreatment allows radicals to he generated
on the spore surface, and/or renders the spore surface
more-susceptible to attack.
Experiments were conducted
to detail the effects of reaction variables, such as tem­
perature, pressure, hydrogen peroxide concentration, expo­
sure time, plasma power, and frequency on the sporicidal
activity observed.
In this way, the optimum combination
of variables with respect to maximizing sporicidal acti­
vity was determined.
vii
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TABLE OF CONTENTS
ACKNOWLEDGEMENT
ABSTRACT
.....................................
v
....................
LIST OF ILLUSTRATIONS
...............................
xii
......................................
xvii
........................................
1
.............................
1
LIST OF TABLES
INTRODUCTION
vi
The Bacterial Spores
Definition of Sterilization.. ......................
2
Current Physical and Chemical Sterilization
Method
.........
3
Current Sterilization Needs.. ......................
9
Interaction of Free Radicals with Bacterial
Spores
.....
9
Oxygen and Hydrogen Radicals Formed by Ionizing
Radiation
••
Methods for Generating FreeRadicals
EXPERIMENTAL SECTION
............
12
................................
16
Microbiological Methods
..........................
Packaging Material and Carriers
Chemicals
9
..................
..........................
16
17
17
Microwave Discharge System •• • • ............
18
Radio Frequency Discharge System
23
.................
• • •
T r * i *i i
V J- .1. _L.
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RESULTS FROM MICROWAVE DISCHARGE
.....................
31
Sporicidal Activity
of Hydrogen Atom
...........
31
Sporicidal Activity
of Hydroxyl Radical ............
32
Sporicidal Activity
of Oxygen Atom
Sporicidal Activity
of Hydroperoxyl Radical
Sporicidal Activity
of •Hj *0; *0Hj and*K02 on
................. 35
.......
Four Bacterial Spores
38
••• 4l
Sporicidal Activity of Water and Microwave
Discharge
........
4.3
Sporicidal Activity of Hydrogen Peroxide and
Microwave Discharge
..............
43
Effect of Carriers on Sporicidal Activity of
Radicals
........................................... 45
Ability of Radicals to Diffuse Through Porous
Polyethylene (Tyvek) Packaging Material
Sporicidal Tests
........
48
................................
48
Wetting Property Tests
............................
CONCLUSION AND DISCUSSION OF MICROWAVE DISCHARGE
RESULTS FROM RF DISCHARGE
50
.....
5^
.............................
56
Temperature Measurement and Control
Effect of Frequency on Temperature
................. 56
.................. 70
Sporicidal Activity of N^O, 02 , H 2> HgO, and HgOg
with RF Discharge at 2.49 MHz with 12 TurnCoil
••• 74
ix
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Effect of Parameters on Sporicidal Activity of
HgOg/Plasma
.......................
Effect of HgOg Pretreatment Time
Effect of HgOg Concentration
79
..............
79
......................
81
*.....
84
....
Effect of Pressure
Effect of Plasma Power
...................
86
Depth of Penetration of H 202 and RF Discharge
System with Paper Disc Substrate
................
88
Effect of Frequency on Sporicidal Activity of
H 202 and RF Discharge
...........................
Dissociation of H202 with RF Discharge
92
......... .....
9^
Sporicidal Activity of HgOg/Plasma on Four
Bacterial Spores
• • •• ........
..............
Comparison between H 202/Plasma and HgOg/Heat
........
97
101
Effect of Plasma Treatment Pressure on
Sporicidal Activity of H 202/Plasma
............
CONCLUSION AND DISCUSSION OF RF DISCHARGE
Temperature Measurement
.............
107
............................
107
Dissociation of H 202 with RF Discharge
Frequency Effect
Pressure Effect
104
..........
108
...................................
108
..........................
109
Sporicidal Activity of HgOg/Plasma with
Anaerobic and Aerobic Spores
Mechanism of Action
•
..................... 110
• • 111
x
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APPENDIX A
............................................
114-
APPENDIX B
..................................... .......
119
REFERENCES
............................................
124
xi
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LIST OF ILLUSTRATIONS
1. Systematic Diagram of Microwave Discharge
19
2. Systematic Diagram of Inductive RF Discharge
24
3. Effect of Temperature and Distance on Sporicidal
Activity of Hydrogen Atom and Hydrogen Gas with
Clostridium sporogenes Spores on Glass Slides
•.. -.
33
4. Effect of the Ratio between Hydrogen and Nitrogen
Dioxide Gases on the Generation of Hydroxyl
Radicals with Bacillus subtilis Spores on
Glass Slides
.•. i.......................
u*... „,,
34
5. Effect of Temperature and Distance on Sporicidal
Activity of Nitrogen Dioxide Gas and Hydroxyl
Radicals with Clostridium sporogenes Spores
On Glass Slides
a
a
a
a
a
a
a
a
a
a
a
a
a
.
a
a
a
a
s
a
a
a
a
a
a
o
a
a
a
a
o
a
e
a
a
36
6 . Effect of Temperature and Distance on Sporicidal
Activity of Oxygen Atom with Bacillus subtilis
Spores on Glass Slides
a..............
yy
7. Effect of Temperature and Distance on Sporicidal
Activity of Oxygen Atom Generated by NgO and
Microwave Discharge with Clostrium sporogenes
Spores on Glass Slides
• • .........................
xi 1
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39
8 . Effect of Temperature and Distance on Sporicidal
Activity of Hydroperoxyl Radical with Clostridium
sporogenes Spores on Glass Slides
.««e... ...........
40
9. Effect of Temperature and Distance on Sporicidal
Activity of Water and Microwave Discharge with
Clostridium sporogenes Spores on Glass Slides
....
44
10. Effect of Temperature and Distance on Sporicidal
Activity of 30$ HgOg with and without Microwave
Discharge on Bacillus suhtilis Spores
.............
46
11. Temperature of 0.5"x0.5"xl.0" Nylon Block Exposed
to RF Plasma at 2.49 MHz Frequency, 1.5 Torr
Pressure and 150 Watts Power
.....................
59
12. Temperature of 0.5"x0.5"xl.0" Stainless Steel,
Polyethylene, and Nylon Blocks Exposed to RF
Plasma at 2.49 MHz Frequency, 1.5 Torr Pressure
and 150 Watts of 1:2 Pulsed Plasma
................
6l
13. Effect of Pressure on Temperature of Nylon Block
Exposed to HgO Plasma at 2.49 MHz Frequency and
150 Watts of 1:2 Pulsed Plasma
..........
63
14. Effect of Power on Temperature of Nylon Block
Exposed to Air Plasma at 2.49 MHz Frequency, 1.5
Torr Pressure and 1:2 Pulsed Plasma
...............
xiii
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64
15. Temperature of Fluoroptic Thermometer Probe and
0.5"x0.5"xl.0" Nylon Block at 1.5 Torr Pressure,
2.49 MHz Frequency and 150 Watts of 1:2 Pulsed
Plasma
...........
65
16. Temperature of Fluoroptic Thermometer Probe
and Nylon Blocks at 2.49 MHz, 1.5 Torr Pressure
and 150 Watts of 1:2 Pulsed Plasma
..........
66
17. Heat History from Differential Scanning
Calorimeter of Polyethylene Microfibers (PEMF)
Samples Treated with 15 Minutes of Air Plasma
at 150 Watts of 1 : 2 Pulsed Power and 1.5
Torr Pressure
•• •
68
18. Temperature of Fluoroptic Thermometer in Air
Plasma at 1.5 Torr Pressure, 2.49 MHz Frequency
and 150 Watts Power
..............................
69
19- Effect of Frequency on Temperature of Fluoroptic
Thermometer Probe and 0.5"x0.5"xl.0" Blocks
after 15 Minutes of 150 Watts of 1:2 Pulsed
Plasma at 1.5 Torr Pressure
............... ..0....
73
20. Effect of Radio Frequency on Temperature of
Fluoroptic Thermometer Probe and 0.5"x0.5"xl.0"
Nylon and Stainless Steel Blocks at 2.49 MHz
(6 Turn Coil), Atmospheric Pressure and 100
Watts of Continuous Plasma
75
xiv
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21. Effect of HgOg Pretreatment Time on Sporicidal
Activity of H 202/Plasma with Tyvek-Packaged
Bacillus subtilis (var. globigii) Spores on
Paper Discs
.......................................
82
22. Effect of H ?0p Concentration on Sporicidal
Activity of H 202/Plasma with Tyvek-Packaged
Bacillus subtilis (var. globigii) Spores on
Paper Discs
...............................
83
23. Effect of Pressure on Sporicidal Activity of
HgOg/Plasma with Tyvek-Packaged Bacillus
subtilis (var. globigii) Spores on Paper
Discs
••
85
2^. Effect of RF Power on Sporicidal Activity of
HgOg/Plasma with Tyvek-Packaged Bacillus
subtilis (var. globigii) Spores on Paper
Discs
................
87
25. Effect of H 202 Concentration and Pretreatment
Time on Sporicidal Activity of H 202/Plasma
with Tyvek-Packaged Bacillus subtilis
(var. globigii)
Spores on Paper Discs
.........
91
26. Effect of Frequency with 12 Turn and 6 Turn
RF Coils on Sporicidal Activity of HgOg and
Plasma with Tyvek-Packaged Bacillus subtilis
(var. globigii)
Spores on Paper Discs
............
AV
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93
2?• Effect of Frequency on Sporicidal Activity
of HgO^/Plasma with Tyvek-Packaged Bacillus
subtilis (var, globigii) Spores on Paper
Discs and the Temperature of the Fluoroptic
Thermometer Probe
.................................
9^
28. Effect of Frequency on Sporicidal Activity
of HgOg/Plasma with Tyvek-Packaged Bacillus
subtilis (var. globigii) Spores on Paper
Discs at a Constant Temperature
...........
96
29. Effect of Plasma Power During H 202 Pretreatment
on Sporicidal Activity of H 2C>2/Plasma with
Tyvek-Packaged Bacillus subtilis (var. globigii)
Spores on Paper Discs
.........
98
xv i
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LIST OF TABLES
1. Sporicidal Activity of «K, *0, »0H, and ‘HOg
with Bi pumilus» B» subtilis, B, subtilis (var.
globigii), and C. sporogenes Spores on Glass
Slides at 60^C, 0.5 Torr Pressure, and 100
Watts Discharge Power for 30 Minutes
42
2. Effect of Carriers on Sporicidal Activity of
HgO/Microwave Discharge with B. subtilis (var.
globigii) Spores at 0.5 Torr Pressure and
100 Watts Discharge for 30 Minutes
................
47
3. Effect of Tyveg Package on Sporicidal
Activity of Radicals with B. subtilis (var.
globigii) Spores on Glass Slides
............
49
4. Effect of Radicals Generated by Microwave
Discharge on Wetting Time of Silk Suture
Loops in 0.05$ P104 Surfactant Solution
after Discharging with 100 Watts Power at
0.5 Torr Pressure for 30 Minutes
..................
52
5. Effect of Pressure on Temperature and Tyvek
Package at 904 KHz Frequency and 200 Watts
for 5 Minutes
......................................
xvii
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58
6 . Physical Properties of RF Coils Used to
Study the Effect of Frequency on Temperature
and Sporicidal Activity
.....
72
7. Sporicidal Activity of Plasma with 0?, N ?0,
and H ? on Tyvek-Packaged B. subtilis (var.
globigii) Spores on Paper Discs
.............. . •••
78
8 . Effect of Power on Sporicidal Activity of
HgO/Plasma with Tyvek-Packaged B. subtilis
(var. globigii) Spores on Paper Discs
.......
80
9. Effect of Vortex Time on Sporicidal Activity
of HgOg/Plasma with Tyvek-Packaged B. subtilis
(var. globigii) Spores
on Paper Discs
...........
89
10. Sporicidal Activity of HgOg/Plasma with
Aerobic and Anaerobic Spores on Paper Discs
inside Tyvek Package
...............................
100
11. Effect of Plasma and Heat Exposure Time on
Sporicidal Activity, HgOg Residuals, and
Temperature Increased of HgOg/Plasma and
H 202/Heat
.........................................
103
12. Effect of Pressure on H 202 Residual, Temperature
and Sporicidal Activity with Tyvek-Packaged
B. subtilis (var. globigii) Spores on Paper
Discs and 15 Minutes of 5.0 mg of H ?02
Pretreatment
••
...........................
xviii
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105
13. Parameters
for
MicrobiologicalWork
....
116
1^. Microbiological Test Results of Data Presented
in Figure 23 - Effectof Pressure
................... 121
15« Microbiological Test Results of Data Presented
in Figure 26 - Effect of Frequency with 12
Turn Coil
• • .....................................
122
16. Microbiological Test Results of Data Presented
in Figure 26 - Effect of Frequency with 6
Turn Coil
....... .................................
123
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INTRODUCTION
The Bacterial Spores
Since their existence was first discovered, a great
deal of research has "been carried out on bacterial spores.
An important property of these spores is their high resis­
tance to attack by various chemical and physical agents.
Therefore, when scientists design methods of achieving ste­
rilization of various medical and pharmaceutical products
and of certain foods they have of necessity been forced to
take this resistance into account.
The most important
spore-formers are members of the genera Bacillus (aerobic
spores) and Clostridium (anaerobic spores).
These two forms
of spores are also the recommended spores by AOAC (Official
Methods of Analysis of the Association of Official Analyti­
cal Chemists) for evaluation of sterilization methods.
The resistance of spores differs within the microbial
population.
Some strains of B. subtilis spores are very
sensitive to heat, whereas others may withstand boiling
temperature for prolonged period of time.
This is also
true with response of spores to chemicals or irradiation
used in their destruction.
Russell
concluded that spores
which are highly resistant to one process are not necessaA
X
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rily insensitive to another process.
This is because
different sterilization methods have different mechanisms
and mode of actions.
Bacterial spores are much more resistant to adverse
effects of heat and chemicals than their corresponding
vegetative cells.
With vegetative cells, the cell wall
functions mainly to limit exchange with the environment
through active transport and maintains the integrity and
rigidity of the cell.
The cell wall offers only limited
protection to detrimental agents.
The core of the spores,
however, is surrounded by a multilayered covering.
Next to
the core is the cell wall surrounded by the cortex and as
many as three spore coats.
Finally there is the expospo-
rium, enveloping all the others.
2
The resistance of
spores has been extensively studied and many publications
have appeared over the years on this subject.
In spite of
these investigations, the nature of the spore and the cause
of its resistance to adverse environmental conditions are
not fully known.
Definition of Sterilization
In a broad sense of the term, sterilization is de­
fined as the use of a physical or chemical procedure to
destroy all microbial life, including highly resistant
"t)3.c"ts2ri3.1 SjP0i?ss■ 3
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3
certainty whether microorganisms of all types have been
killed because we may not even be aware of the existence
of some species or have media suitable for culturing all
organisms, it becomes more practical, as pointed out by
Ll
Bruch and Bruch,
rilization.
to employ a process definition of ste­
By this definition, sterilization is the pro­
cess by which living organisms are removed or killed to
the extent that they are no longer detectable in standard
culture media in which they previously have been found to
proliferate.
According to this definition, both the pro­
cess used to achieve sterility and the methods for testing
for it are equally important.
Current Physical and Chemical Sterilization Method
Various methods have been proposed or used for steri­
lization, for example: heat, radiation, filtration, cold,
desiccation, cellular disintegration, and chemical gases
and v a p o r . H o w e v e r , in recent years the sterilization of
different types of articles such as medical equipments,
plastic goods, foods, and raw materials has commonly in­
volved only four types of methods - heat (dry heat and
steam), liquid chemical (glutaraldehyde and chlorine di­
oxide), radiation (Co 60 and electron beam), and gas
(ethylene oxide).
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Heat (dry heat and steam)
Heat is the oldest and most recognized agent of des­
truction.
Moist and dry heat are classic sterilizing media.
Steam under pressure is inexpensive and sterilizes pene­
trable materials and exposed surfaces rapidly.
Thermal
death of microorganisms exposed to steam takes place as the
result of the inactivation of essential cellular proteins
or enzymes by denaturation.
Exposure to steam at 132°C and
2200 mmHg pressure for five minutes will inactivate the
most thermal resistant spores.
In contrast, the mechanism
for destruction with dry heat is primarily an oxidative
process, and temperature of 170°C for 60 minutes are re­
quired for inactivation of resistant spores.
While wet or
dry heat at sufficiently high temperature is effective for
sterilization, its use is restricted to those articles
which can remain undamaged by the temperature required.
Thus large classes of temperature or moisture sensitive
articles cannot be sterilized in this manner.
Radiation (Co 60 and electron beam)
Cobalt-60 gamma rays are mainly used for the large
scale sterilization of manufactured items, but has found
limited use in hospitals.
This is because the gamma rays
are highly penetrating and expensive facilities are re­
quired for handling and shielding the radio-active source.
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c
J
Its use for industrial sterilization has been increasing
worldwide for the following reasons:
(a) Radiation causes little or no deterioration of most
material employed for single-use medical and hos­
pital products.
(b) It is a simple and reliable process, which is easy
to control and maintain in good operating condition.
(c) Because of the great penetrating ability of gamma
rays, products in their sealed, final shipping con­
tainers may be sterilized.
Studies clearly indicate that deoxyribonucleic acid
(DNA) is a prime target of ionizing radiation destruction
of microorganisms.
This target may be altered either
directly by ionizing radiation or indirectly by the action
of free radicals produced by an ionization event elsewhere
in the system.
This can result in either single or
double strand breaks,
7
the formation of a number of
different nucleotide dimer forms,
8
and DNA degradation.
Q
Among the disadvantage are the possible degradation of
some plastics and changes occurring in their bulk and
surface properties.
The irradiation of polymers results
in either crosslinking or degradation depending on the
chemical structure of the polymers.
10
Electron beam has gained increased usage in the
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past few years as a method of industrial sterilization
as the instrumentation required to accelerate the elec­
tron have become more reliable.
Electron beam has the
disadvantages of requiring expensive instrumentation and
possessing limited penetration abilities.
Liquid Chemical (glutaraldehyde and chlorine dioxide)
Aqueous 2% solutions of glutaraldehyde have been
employed mainly for the sterilization of medical and other
materials that cannot be sterilized by heat or irradiation.
Advantages of liquid glutaraldehyde as a chemosterilant
include:
(a) effective broad spectrum sporicidal activity;
(b) active in the presence of organic matter;
(c) noncorrosive action toward metal, rubber, lenses,
and most materials;
(d) ease of use and relative lack of toxicity.
The chemistry of the reaction of glutaraldehyde
with protein has not been definitely elucidated.
It is
likely that several reactions occur, giving rise to a
number of products.
The reaction of free aldehyde with
a primary amine of the protein is followed by conden­
sation of additional free glutaraldehyde and leads to
the formation of a l,3 »^i5-substituded pyridinium salt.
This mechanism ties in with the observation of a new
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absorption peak at 265 ran as proteins are crosslinked.
11
The pyridinium linkage is not the only type
of crosslink in glutaraldehyde-treated proteins, but it
is thought to represent a significant contribution to
the chemistry of glutaraldehyde sterilization.
The
drawbacks with glutaraldehyde solution are long steri­
lization times (10-12 hours is recommended), and the
fact that they cannot be used on packaged items.
More recently, aqueous solutions of chlorine di­
oxide have been used as a liquid chemical sterilant.
This solution is reported to exhibit more rapid sporicidal activity than glutaraldehyde, but appears to be less
compatible with medical products, frequently discoloring
these materials.
The mode of inactivation of bacterial
spores with chlorine dioxide is unknown, but the chemi­
cal is known to react rapidly with phenolic and sulfhydryl groups.
Gas (ethylene oxide)
Sterilization using gaseous agents has been prac­
ticed for many years.
The most widely used gaseous
sterilizing agent is ethylene oxide (ETO).
ETO has been
shown to have a number of medical applications.
12
This
gas is used in hospitals in the United States almost
exclusively to sterilize items that cannot be sterilized
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
by steam.
Experimental evidence indicates that alkyla-
tion is the main reaction of ETO with nucleic acids which
cause injury and/or death of spores or vegetative cells.
The turnaround time for ETO sterilization is approxima­
tely 12 hours._ This includes Z - k hours for steriliza­
tion and at least 8 hours for aeration.
Although ethylene oxide gas permits effective
sterilization, the toxic residue formed by the ETO in
the material to be sterilized is a problema
ETO is
toxic, mutagenic, carcinogenic, and irritating to eyes
and mucous membranes.
Because it is highly penetrating,
this gas can leave a residue that must be removed by
mechanical ventilation.
Acknowledging the danger, the
Occupational Safety and Health Administration (OSHA),
in the April 21, 1983, Federal Register, proposed that
the permissible exposure limits for ETO be reduced sig­
nificantly - from an eight-hour time-weighted average
of 50 parts per million (ppm) to 1 ppm.
13
It is appa­
rent, therefore, that ETO sterilization became widely
used not because it is an ideal process, but rather
since there seemed to be no alternative gas sterilant
method which is capable of as fast a sporicidal action
without any drawbacks from the toxicological or envi­
ronmental viewpoint.
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Current Sterilization Needs
Because of the disadvantages associated with current
low temperature sterilization processes, a new low tempera­
ture process that has rapid sporicidal activity and does
not leave any toxic residual would "be of considerable com­
mercial interest.
Since liquid sterilants cannot he used
on packaged items, a gas process would he required.
Since
the ideal sterilization process requires rapid sporicidal
action at low temperature without any toxic residual, a
sterilization system hased on free radical or radical-ion
chemistry would appear to be a logic choice.
Interaction of Free Radicals with Bacterial Spores
The objective of this research was to investigate
the interaction of free radicals with anaerobic and aerobic
bacterials.
The free radicals selected for investigation
were those considered to be of primary importance in the
inactivation of organisms exposed to ionizing radiation.
As a result of this work, a better understanding should be
obtained of the ability of specific free radical to inac­
tivate both anaerobic and aerobic bacterial spores.
Oxvgen and Hydrogen Radicals Formed by Ionizing Radiation
Radiation inactivation of biologically active pro­
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10
teins in aqueous system is usually attributed to both
reducing (*H, e” ) and oxidizing (»0H) radical species.
Studies by Grecz and Schgal
16
with botulinum toxin, a model
protein molecule found in C. botulinum spores, showed that
radiation inactivation of this molecule was accomplished
to 48% by *H, 14$ by e” , 31
aq
by *0H, and 7 % "by direct hits.
Friedman and G r e c z ^ used 2M ethanol (a scavenger of
•OH radicals) and 1M sodium nitrate (a scavenger of *H
radicals and hydrated electrons) and proposed that radia­
tion inactivation of spores at temperature below 50°C is
due to DNA damage inflicted by «0H radicals whereas spores
death above 50°C seems to involve protein (enzyme) inacti­
vation due to a combination action of heat and reducing
(*H and e~ ) as well as oxidizing (*0H) radical species,
aq
Nitrous oxide (NgO) produces radiosensitization in bacte­
rial spores, but this can be completely reversed by tertbutanol.
NgO may act by scavenging solvated electrons in
the presence of water and increasing the yield of
-
Besides *H, e , and *0H, Feldberg and Carew
aq
20
also
included the superoxide anion radical (*0^) and its protonated hydroperoxyl radical (‘HC^) as protein-recognizable
active species in gamma-irradiated aqueous solution.
Ewing
21
found an aqueous suspensions of bacterial spores,
irradiated in air, show an increased sensitivity when they
are subsequently irradiated under anoxic condition^
This
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sensitization is experimentally attributed to the action
of
formed during the x-ray exposure in air.
However,
H 202 formed in this way cannot function as an anoxic spore
sensitizer if 10
M tert-butanol is present.
These results
emphasize that a synergistic relationship exists between *0H
radicals and HgOg.
Also, the radiation sensitization by
NgO requires that both H 202 and *0H radicals be present.22
One explanation for the synergistic relationship between
H 202 and «0H radicals he concluded
23 24
is the formation of
•0“ (and/or •HOO, the product of such a reaction, which is
likely the agent that actually causes damage.
Certain observations2-’"'50 suggest that superoxide
toxicity is the result of direct or indirect attack at the
molecular level by superoxide or secondarily generated
radicals.
The reaction *02 + H 202 —
-+ 02 + *0H + OH
pro­
vides a mechanism for converting the relatively unreactive
superoxide ion radical into the potently oxidizing active
hydroxyl radical.
Synergistic effects of H 202 and ultra­
violet irradiation has been found to occur-^"-5^ and it is
believed that spores and non-sporing bacteria are destroyed
by the »0H formed by decomposition of HgOg.
Apparently, hydrogen radical (»H), hydroxyl radical
(•OH), superoxide radical (*02), and hydroperoxyl radical
(*H02) are all sporicidal active species.
However, the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
12
hydroxyl radical is capable of producing broad, nonspecific
oxidative damage, as well as initiating free radical chain
reactions, and a number of investigators have suggested
that it is biologically the most damaging species.35-^2
In this research the ability of *0H, ’HOg,
*H, and *0
to inactivate both anaerobic and aerobic bacterial spores
will be investigated.
Methods for Generating Free Radicals
lj.3
There are several ways ^ to generate the hydrogen,
oxygen, hydroxyl, and hydroperoxyl radicals in the gas
phase.
Only low temperature processes are discussed.
Electrical Dis c h a r g e ^ In electrical discharges the energy necessary for
dissociating molecules of the gas or vapor is supplied pri­
marily by electron collisions; dissociation into radicals
is the result of complex interactions between electrons,
ions, and molecules.
Microwave discharge and radio fre­
quency (RF) discharges were used in this work.
Microwave Discharge.
Hydrogen atoms can be easily
generated by direct discharge through hydrogen gas.
The
hydroxyl radicals can be generated by the reaction between
hydrogen atom and nitrogen dioxide (N02 ) gas.
By flowing
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
hh,
HgO vapor through the discharge,
hydrogen atoms and
hydroxyl radicals can be generated simutaneously.
iie
Venugopalan and Shih J concluded from their work
with low-pressure microwave cavity discharge of hydroSt
gen peroxide vapor that «H and *0H were produced in
proportions which varied with the applied power.
Even
at the lowest power density H 2C>2 was not found in a
cold trap at 195°K, suggesting that either the dissoci­
ation of H 202 was complete or the *0H radicals produced
by scission of H 202 in the plasma reacted rapidly with
undissociated H 202 to produce *HC>2 radicals and H 20
The *H02 radicals had been identified in their system
mass spectrometricallyo
But the *H02 radicals will be
removed by a fast reaction with »0H radicals to form
HgO and 02 =
Hydroperoxyl radicals can also be generated
by the reaction between hydrogen atom and oxygen gas
with He, Ar, or HgO as the third b o d y . ^
Microwave discharge is a very convenient method to
study the sporicidal activity of various radicals.
In
addition, the effects of temperature, distance, exposure
time, and package on sporicidal activity of radicals can
also be determined with microwave discharge.
RF Discharge.
Low temperature RF discharge was
used in the current research for a combination of factors,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
14
i.e., ease of control, simplicity of construction,
greater stability, and probably most important, a
greater purity of the discharge products because this
method does not involve metallic electrodes in contact
with the discharge gases.
Although sterilization
claims have been made for low temperature RF discharges,
J?o-o3 nQ systematic study has ever been published on
the use of RF discharges as a method of sterilization.
Discharge through hydrogen gas and water vapor
is a very good method to produce hydrogen and hydroxyl
radicals.
Oldenberg and Frost-3
found from their dis­
charge system that after interrupting the discharge
through HgOg the absorption spectrum of *0H radicals
disappeared at a faster rate than in the discharge
through'H20.
In HgO the absorption of *0H radicals
could be traced as long as 0.4 sec., but in HgOg under
similar conditions it was much fainter and disappeared
within l/lOO sec.
That *0H is certainly produced in the
discharge is shown by the fact that the •OH emission
from the discharge through HgOg was even stronger than
the *0H emission from the HgO discharge under the same
condition of pressure and power.
Since the main com­
ponent of the HgOg/aischarge consists of HgOg, they
concluded that the rapid disappearance of .OH is cause
by a bimolecular reaction with H«0„ to produce *H0„
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
radicals.
*57
Foner^' found that discharge products from HgOg
observed within 1 millisecond (ms) of H 202 decomposition
are *H, H 2 , *0, *0H, 02 , *HC>2 , HgOg, HgO and Oy, they com­
prise a complete catalog of the known components in the
hydrogen-oxygen system.
Within a few milliseconds the
system becomes considerably simplified, the predominant
radical component being the hydroperoxyl radical.
Ultraviolet Ra d i a t i o n ^ -^
The photolysis of HgO and H 202 vapors provides a con­
venient source of hydrogen, hydroxyl and hydroperoxyl radi­
cals.
However, preliminary studies using ultraviolet radi­
ation revealed no apparent advantages over microwave or RF
discharge and was not pursued futher in this research.
Ionizing Radiation
Due to the short wavelength of the ionizing radiation,
such as x-rays and gamma-rays,
*H, *0H and *H02 radicals
are easily produced from H 2 gas and HgO and H 202 vapors.
ionizing radiation with enough energy to have good
penetration, however, requires expensive equipment or in­
volves radio-active elements which require special shiel­
ding.
As stated earlier, the major purpose of the current
work was to attempt to develop an alternative to ioniza­
tion sterilization, particularly one which might ultimately
be used with T>ackaged materials ?
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EXPERIMENTAL SECTION
Microbiological Methods
Since vegetative organisms will die under vacuum con­
dition and thus confuse the test results, only spores were
used in this research.
Four types of spores were included
in these studies ;
1. Clostridium sporogenes ATCC 3584 (American Type Culture
Collection).
This is a typical anaerobic spore recom­
mended by AOAC to evaluate the efficacy of chemicals as
sterilizing agents.
2. Bacillus subtilis ATCC 19659*
This is a typical aerobic
spore recommended by AOAC to evaluate the efficacy of
chemicals as sterilizing agents.
3* Bacillus subtilis (var. globigii) ATCC 9372.
An aerobic
spore which is hydrogen peroxide resistant and the bio­
logical indicator for ETO sterilizer.
4. Bacillus numilus ATCC 27142.
An aerobic spores which is
radiation resistant and thus is used as the biological
indicator for gamma radiation.
All the microbiological work including preparation
of stock cultures, inoculation of substrates and assay for
survivors of the spores are described in Appendix A.
The
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reproducibility of the microbiological test results is
discussed in Appendix B.
Packaging Material and Carriers
Some samples were sealed inside spunbounded polyethy­
lene (Tyvek) packages.
The Tyvek packaging had a maximum
pore size diameter of 10 microns as determined by bubble
point measurement.
Tyvek, which is constructed of 2.8
microns average diamentr fibers, has a softening point of
125°C and a melting point of 130°C.
With the packaged
substrates, the relative ability of various radicals gene­
rated outside the package to diffuse through the porous
packaging material and deactivate the spores inside the
package can be determined.
The biological indicators for both ETO and gamma
radiation use paper spore strips as the carriers for the
test organisms.
Therefore, paper discs (Schleicher &
Schnell #7^-0-E with t ” circles) which have the same proper­
ties as the spore strips were used as the major carrier
for the efficacy test.
One inch square glass slides were
also used in the microwave discharge studies.
Chemicals
The following is a list of all the chemicals used in
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
*
10
A
this research.
All the gases were obtained from Kennedy
Welding Supply Corp., Arlington, Texas and reagents were
supplied from J. T. Backer Chemical Co., unless otherwise
noted below.
(1) Argon (Ar)s
9 9 .6$.
(2) Carbon Dioxide (COg):
/o \
T T - i i , -
\ 3 j nt!xj.uiu
/ tr — % ,
r\r\
99*7$*
r\r\r\tff
yy*yyy/°>
(^) Hydrogen (H^)*
99*999$.
(5) Hydrogen Peroxide (HgOg):
30$, Superoxol.
Other con­
centration of HgOg were prepared by diluting the 30$
HgOg with deionized water.
(6 ) Nitrogen (N2 ):
99*999$*
(7) Nitrogen Dioxide (N02):
(8 ) Nitrous Oxide (NgO):
(9) Oxygen (02):
(10) P104:
99*5$*
99*5$*
99*99$
Pluronic surfactant from BASF Wyandotte Corp.
(11) Potassium Iodide (KI)s
meet A.C.S. specification.
(12) Sulfuric Acid (HgSO^):
meet A.C.S. specification.
(13) Water (HgO):
deionized water.
Microwave Discharge System
The apparatus used for the microwave discharge study
is shown in Figure 1.
The components are listed as
follows:
(1) Raytheon PGM-10X1 microwave power generator which pro-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
liquid
19
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
duce 10 to 100 watts of microwave energy at 2.45 GHz.
(2) Coaxial cavity:
for coupling output of microwave
generator to plasma generating system.
(3) Teledyne Hastings-Raydist vacuum gauge tube type DV-4D
(4) Teledyne Hastings-Raydist Thermocouple vacuum gauge
model VT-5A.
(5) Kontes K —847000-15 nigh vacuum stopcock.
(6 ) Kontes K-926250-25 two piece vacuum trap.
(7) Sargent-welch two-stage DuoSeal vacuum pump model 1397.
(8) Leybold Heraeus type PS111A pressure switch.
(9) Leybold Heraeus type 28121 electromagnetic right angle
valve.
(10) Leybold Heraeus type SV110 switching amplifier.
(11) 0-ring.
(12) Chamber door.
(13) Ground spherical ball and socket joint.
(14) High vacuum Telfon valve used to release vacuum.
(15) Standard taper socket joint; an opening for either 16
or 1 7 .
(16) Two pieces of glass tubing, with filter stick porous
tip on one piece, connected together with Tygon
tubing; can be used through opening 15 to introduce
the second gas or vapor to react with the radicals
generated by microwave discharge.
(17) Glass stopper used to close the opening 15*
(18) Pyrex reaction chamber with 6 " I.D. and 43" long.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
21
(19) Whitey micro-metering valve used to control the flow
of gas.
(20) Buret with 5 ml capacity and 1/100 ml subdivisions
used to deliver the liquid.
(21) Teflon varibor stopcock, same as Kontes K-811110221 but with capillary tubing on bothside;
used to
control the flow of liquid.
(22) Capillary tube.
(23) 60°C water bath.
(2*0
Ground spherical socket joint.
(25)
Ground spherical ball joint.
(26) Small piece of glass wool between capillary tube and
the three-neck round bottom flask.
(27) Heating tape used to control the temperature.
All the tests were conducted with a flow-through
system.
Initially, gas or vapor was first allowed to flow
through the discharge tube.
The selected microwave power
was then applied to the discharge cavity.
For a gas dis­
charge, the pressure of the chamber was controlled by the
flow rate of the gas with the electromagnetic right angle
valve (No. 9 of Figure 1) at the close position.
For a
vapor discharge, the chamber was first evacuated through
the main vacuum line with the stopcock (No. 5 of Figure 1)
at the open position, and then the pressure was controlled
by the electromagnetic right angle valve with the stopcock
Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission.
C.C.
rs a
at the closed position.
Microwave discharge is a clean method to study the
sporicidal activity of radicals, because radicals are the
only species generated in the microwave discharges
With
the above described experimental set up. the following
effects on sporicidal activity were studied:
(1) Effect of Distance:
The radicals generated by the
microwave discharge need to flow to the site of the
spores in order to achieve any sporicidal activity.
Samples were located at 5"» 15"» and 25" from the
discharge cavity to determine the effect of distance
on sporicidal activity.
(2) Effect of Temperature;
The rates of most chemical
reactions increase with increasing temperature.
The
temperature also increases the germicidal activity of
most germicidal agents.
Since radicals are very active
species, the effect of temperature on the sporicidal
activity was of interest.
Studys were conducted at
room temperature (20-24°C) and 60°C.
(3) Effect of Package:
To determine whether radicals gene­
rated outside a package., can penetrate the packaging
material and deactivate spores, sporicidal testing
was conducted inside sealed packages.
(^) Effect of Radical Type:
The hydrogen atom was generated
by flowing the Hg gas through the discharge cavity.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
23
The hydroxyl radical was generated by the reaction of
hydrogen atom and nitrogen dioxide gas.
The oxygen
atom was generated by flowing the oxygen, nitrous
oxide, or carbon dioxide gas through the discharge
cavity.
The hydroperoxyl radical was generated by the
r
o p n + n Ar»
-l. \*CL l*
r>-P
V>^r/^y»r\cr^v> CL
p +vrs^xn
vm
_L AXJf
the third body.
p
Ccxx^
ovtrcrovi1
era a
urn
+ Vi wn pu +w wcjr.
« * X v/ii
p c*
CLd
Microwave discharges with water and
hydrogen peroxide vapors were also studied.
In the
absence of suitable spectroscopic identification me­
thods, it was not possible to identify radicals and
radical concentrations spectroscopically.
Verifi­
cation that the assigned radicals were generated are
based on the following considerations:
(a) Well documented methods for generating specific
radicals were used.
(b) Different sporicidal activity was observed with
different reactions.
(c) Different reactions showed different temperature
and distance effects on sporicidal activity.
Radio Frequency Discharge System
The complete inductive radio frequency discharge
system for this research is shown in Figure 2.
A detailed
description of all components are listed below.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
24
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(1) Hewlett Packard (HP) model 8116A pulse/function genera­
tor.
(2) Electronic Navigation Industries (ENI) model A1000 RF
power amplifier.
(3) Bird Electronic Corp. wattmeter model **410.
(*)■) Cardwell Condenser Corp. model 152-0002-001 variable air
capacitors.
(5) Belden 8259 RG-58 A/u cable.
(6 ) Olympic Wire & Cable Corp. RG
8/u cable.
(7) RF coil made of 5 " O.D. copper tubing.
(8) ground
(9) Pyrex reaction chamber with 6" I.D. and 18" long
(10) Sargent-Welch two-stage DuoSeal vacuum pump model 1397(11) Kontes K -926250 two piece vacuum trap.
(12) M S
type 253A-1-40-1 throttling valve.
(13) M S
type 252A throttling valve controller with 4 set-
points and valve position options.
(1*0 M S
type PDR-D-1 power supply and digital
readout unit.
(15) M S
type 222B absolute transducer with 10 mmHg full
scale pressure range.
(16) Kontes K-8*J-7000-15 high vacuum stopcock.
(17) Luxtron model 1000A Fluoroptic thermometer.
(18) Luxtron type LSA-*J-m optical fiber probe.
(19) Luxtron type VFT-10 vacuum/pressure feedthrough.
(20) Red sleeve serum rubber stopper with needle septum for
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
liquid injection.
(21) Whitey micro-metering valve.
This RF discharge system has several special charac­
teristics which make the system unique and easy to control.
(1) Pressure Control?
With ^ set-points MKS pressure con­
troller, the pressure of the chamber can be easily
changed and precisely controlled.
(2) Frequency Control;
Most of the RF generators operate
at a frequency of 13.56 MHz, a prescribed frequency of
operation by the Federal Communications Commission, to
prevent interference with other communication bands.
If the experiments are carried out in a Faraday cage
(a grounded copper mesh screen), any frequency may be
used.
In order to study the possible effects of fre­
quency on sporicidal activity, a wide frequency range
function generator and RF power amplifier were used,
along with the Faraday cage to minimize stray radia­
tion.
Optimization of coupling the RF energy into the
discharge is done by matching the gas load impedance to
the impedance of the amplifier of the RF source.
Impe­
dance matching is achieved by a tuning process with
the capacitors.
Once tuned, the matched circuit is in
a resistor-inductor-capacitor resonance circuit.
The
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
frequency at which resonance occurs can he expressed as
f
= l/(2]tjLC).
A range of frequencies can therefore
he obtained with a given coil hy using a matching net­
work that consists of variable air capacitors.
(3) Power Pulsed Discharge:
The function generator used in
the present process may he continuous or pulsed.
That
is, the power may he applied continuously to the plasma
or the plasma may he pulsed hy activiting the plasma in
a cyclic manner while maintaining the pressure of the
plasma constant.
The use of a pulsed plasma prevents
the overheating of the gas within the chamber as well as
preventing the overheating of objects that need to he
studied,
(*0 Temperature Measurement:
Translational temperature is
the temperature used to describe the gas temperature of
plasma.
74-76
The common method of measuring transla­
tional temperature uses metallic wire or thermocouples.
This method cannot he used in a plasma environment due
to the presence of high voltage and RF fields that can
produce eddy current heating or noise pick-up in the
sensor, accidental shorting and/or spark conducting hy
the sensor. An optically based "Fluoroptic" thermometer
was used in this research and has been found to yield
precise measurement of temperature in electrically
hostile environment. 7 7 * 7 &
Fluoroptic thermometers are
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
not limited to line-of-sight measurements of surface
temperatures.
The probe can be inserted into solid
materials to monitor the temperature of objects exposed
to the plasma.
This device utilizes the physical phenomenon that
rare earth phosphores, when excited, displays a ratio
of intensities from selected spectral lines which vary
with the temperature of the phosphore.
In this device,
a small amount of phosphor is bound to the tip of an
optical fiber.
Ultraviolet light is transmitted from
the fiber to excite the phosphor, and then the resulting
fluoroscence is transmitted back to the control electro­
nics through the same fiber.
The Fluoroptic optical
fiber probe contains no metal or conductors, and is thus
electrically isolated from the measurement environment.
In the presence of an alternating electromagnetic field,
it does not suffer from eddy heating or noise pick-up.
It can therefore be used to make measurements in electro­
static fields without disturbing normal component opera­
tion.
The Fluoroptic thermometer appears to provide the
best method of measuring the translational temperature of
plasma and the temperature of objects exposed to the
plasma.
The Fluoroptic thermometer provided a very good
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
?Q
S
~
method of measuring the temperature of objects exposed to
an RF plasma as long as the object was large enough to
provide easy contact with the thermometer probe.
The tem­
perature of small objects (such as fibers) could not be
determined directly by the Fluoroptic thermometer.
There­
fore, tests were conducted with polyethylene microfibers
(2-5 micron diameters) using a Perkin-Eimer DSC-2 Differen­
tial Scanning Calorimeter to determine the thermal history
of the polyethylene microfibers after exposure to RF
plasma.
The Differential Scanning Calorimeter is a sophi­
sticated instrument for the measurement and characteriza­
tion of the thermal properties of materials.
When a tran­
sition such as melting or crystallization occurs in the
sample material, an endothermic or exothermic reaction
takes place.
The change in power required to maintain the
sample holder at the same temperature as the reference
holder during the transition is recorded as a peak.
Endo­
thermic transitions are represented by upscale departures
from the ordinate baseline and exothermic reactions are
represented by downscale departures from the baseline.
Many polymer classes are well known for their tendency
to crystallize on heating.
If the material was held at a
certain temperature, crystal growth would be encouraged
in that region and a transition peak at that temperature
will appear on the melting curve.
Since higher temperature
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
30
will wipe out the lower thermal history, only the highest
thermal history will show up on heating.
The thermal
history can easily he identified hy rescaning the sample.
If the rescan still shows the transition then the peak is
not a thermal history.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
RESULTS FROM MICROWAVE DISCHARGE
In all the test results, the sporicidal activity is
expressed as the ratio of the number surviving the test
(S) to the initial number of spores which were placed on
the specimen prior to the test ( .
thus vary between zero and one.
The ratio of S/S^ can
The smaller the ratio, the
better the sporicidal activity.
In addition to the specified conditions in each test,
all experiments were conducted at 0.5 torr pressure and
100 watts discharge power.
minutes.
All tests were run for 30
Glass slides were used as the carrier for the
test organisms.
Approximately 10^ spores were inoculated
onto one side of the glass slide which was placed hori­
zontally in the center of the chamber with the spore side
facing up.
The flow rates of the liquid were set at 0.1
ml/minute.
The sporicidal activity of the various radicals
and the effects of distance, temperature, and Tyvek packa­
ging are presented below.
Sporicidal Activity of Hydrogen Atom (*H)
The hydrogen atom was generated by flowing the E ^ gas
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
through the discharge cavity.
The sporicidal activity of
hydrogen gas and hydrogen atom on C. sporogenes spores are
presented in Figure 3«
No appreciable sporicidal activity
was noted with H ? gas at room temperature and slight spori­
cidal activity was observed at 60°C.
The sporicidal acti­
vity of the hydrogen atom increases with increasing tempe­
rature and decreases when the distance from the discharge
was increased from 5 ” to 15” .
At greater distances the
sporicidal activity of the system appears to level off.
Sporicidal Activity of Hydroxyl Radical (»0H)
The hydroxyl radical was generated by the reaction of
hydrogen atom with nitrogen dioxide.
The hydrogen atom was
generated by the microwave discharge of hydrogen gas.
The
nitrogen dioxide gas was introduced through the gas inlet
port labeled 15 in Figure 1.
Tests with different ratios
of hydrogen gas and nitrogen dioxide gas were first con­
ducted to determine the condition for producing maximum
concentrations of hydroxyl radical.
The- test results at
6o°C on B. subtilis spores showed (Figure 4) that a higher
partial pressure of NOg to H ^ apparently produced greater
quantities of hydroxyl radical as determined by the in­
creased sporicidal activity,
A ratio of 0.1 torr of Hg to
0.4 torr of NOg produced maximum sporicidal activity.
Hydroxyl radicals in all subsequent tests were produced at
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
33
1x10 -1
IxlO-2
c
s/s
' o
' IxlO-3!
1x 10
-4
a : H 2 at room temp
d
b : H 0 at 60°C
c : *H at room temp,
1x10-5!
d : -H at 60°C
5
10
15
20
25
Distance from Discharge Cavity (inches)
Figure 3. Effect of Temperature and Distance on
Sporicidal Activity of Hydrogen Atom
and Hydrogen Gas with Clostridium
soorogenes Spores on Glass Slides
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3^
1x10 -1
a
1x 10
s /s,
1x10 -3
Partial Pressure
(torr)
1x10
H,
NO,
a :
0.3
0.2
b :
0.2
0.3
c :
0.1
O A
-4
1x 10 -5
5
10
15
20
25
Distance from Discharge Cavity (inches)
Figure
Effect of the Ratio between Hydrogen and
Nitrogen Dioxide Gases on the Generation
of Hydroxyl Radical with Bacillus subtilis
Spores on Glass Slides
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
35
this ratio of H 2 to NOg.
The sporicidal activity of hydroxyl radicals and
nitrogen dioxide gas on C. sporogenes spores are presented
in Figure 5=
Negligible kill was noted with N02 alone at
room temperature and slight activity was observed at 60°CS
Higher temperature had no significant effect on the spori­
cidal activity of hydroxyl radicals.
A more dramatic dis­
tance effect was observed with hydroxyl radicals than
hydrogen atoms.
Hydroxyl radicals appear to have better
sporicidal activity than hydrogen atoms at room temperature
and about the same activity as hydrogen atoms at 60°C.
Hydrogen atoms can exhibit sporicidal activity at a greater
distance from the discharge than hydroxyl radicals, which
is consistent with the hydrogen atom being the least reac­
tive of the two radicals.
Sporicidal Activity of Oxygen Atom (*0)
Oxygen, carbon dioxide, and nitrous oxide gases were
used with microwave discharge to generate the oxygen atom
at room temperature and 60°C.
The results on B. subtilis
spores are presented in Figure 6 .
The best sporicidal
activity of oxygen atoms was obtained with N20 gas.
This
is probably because NgO has the lowest bond dissociation
energy to generate oxygen atom, i.e., 4-0 kcal/mole for NgO,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
36
l 4-
1x10 -1
1x10
/
-2
//
s/s,
1x10 -3
b : no2 at
ON
o
o
o
a : no2 at room
1x10
c : •OH at room
1x10 -5 ..
--------—
d : •OH at
6o°C
4----------- 1--------- — I----------- 1----------- 1_
5
10
15
20
25
Distance from Discharge Cavity (inches)
Figure 5* Effect of Temperature and Distance on
Sporicidal Activity of Nitrogen Dioxide
Gas and Hydroxyl Radical with Clostridium
sporogenes Spores on Glass Slides
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
37
1x10
-1
IxlO-2 ■■
-1x10-3
a : O^/discharge at room temp,
1x10
1x10 -5
..
1
5
b
:02/discharge at 6o°C
c
:C02/discharge at
room temp,
d
:NgO/discharge at
room temp,
e
:N,0/discharge at
60°C
1
10
1
15
1
(--------------- —
20
25
Distance from Discharge Cavity (inches)
Figure 6 . Effect of Temperature and Distance
on Sporicidal Activity of Oxygen
Atom with Bacillus subtilis Spores
on Glass Slides
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
38
127.2 kcal/mole for C02 , and 118.3 kcal/ml for 02 »
The
least sporicidal activity was obtained with 02 gas.
This
is probably due to the rapid reaction of oxygen atom and
oxygen gas to form ozone, and the low sporicidal activity
of ozone compare to oxygen atom.
Apparently, the good
sporicidal activity of the oxygen atom from NgO is not only
because of the low bond dissociation energy, but also be­
cause there are no other fast reactions to scavenge the
oxygen atom.
The sporicidal activity of oxygen atoms
generated by N20 and microwave discharge with C. sporogenes
spores is shown in Figure 7.
In general, no significant
temperature effect was noted, but a definite distance
effect was observed with the oxygen atom generated by NgO
and microwave discharge.
Sporicidal Activity of Hvdroperoxyl Radical (’HCU)
The hydroperoxyl radical was generated by the reaction
of hydrogen atom and oxygen gas with water as the third
body at a partial pressure of 0.1 torr of Hg, 0.2 torr of
02, and 0.2 torr of H 20.
The sporicidal activity of hydro­
peroxyl radical on C. sporogenes spores are presented in
Figure 8 .
The sporicidal activity increases with increasing
temperature and decreases with increasing distance from the
discharge.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
39
1
1x10
1x10
1x10
1
-2
room
temp.
■4
5
10
15
20
25
Distance from Discharge Cavity (inches)
Figure 7. Effect of Temperature and Distance on
Sporicidal Activity of Oxygen Atom
Generated "by NgO and Microwave Discharge
with Clostridium sporogenes Spores on
Glass Slides
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
kn
1x10
room
•temp
1x10
-2
o
1x10
60°C
-k
1x10 * ■
1x10
5
10
15
20
25
Distance from Discharge Cavity (inches)
Figure 8 . Effect of Temperature and Distance
on Sporicidal Activity of Hydroperoxyl
Radical with Clostridium snorogenes
Spores on Glass Slides
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4i
Unfortunately, no instrumental methods were available
to detect whether the hydroperoxyl radical was generated.
By comparing the sporicidal activity and the effects of
temperature and distance to other radicals, it is believed
that a different sporicidal species was generated.
Accor­
ding to the literature-^, the most likely species would be
hydroperoxyl radical.
Sporicidal Activity of «H. *0. *0H, and ‘HCU
on Four Bacterial Spores
B. pumilus, B. subtilis (var. globigii), B. subtilis,
and C. sporogenes spores were used with hydrogen, oxygen,
hydroxyl, and hydroperoxyl radicals to evaluate the spori­
cidal activity of these four active species on four diffe­
rent bacterial spores.
In this manner, both the bacterial
spores most resistant to radicals and the most sporicidally
radical species could be determined.
All the radicals were
produced by the methods previously noted.
All tests were
run with one sample located 5 " away from the discharge
cavity.
All tests were conducted at 0.5 torr pressure and
6o°C for 30 minutes.
The results of this study (Table 1) show that C.
sporogenes, an anaerobic spore, was the easiest to kill
under the test conditions.
Of the aerobic spores tested,
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE 1
SPORICIDAL ACTIVITY OF *H, *0, «0H, AND »HOg WITH B. PUMII.US. B, SUBTILIS.•'
B. SUBTILIS (VAR. GLOBIGIl), AND C. SPOROGENES SPORES ON GLASS SLIDES AT
60°C, 0.5 TORR PRESSURE, AND 100 WATTS DISCHARGE POWER FOR 30 MINUTES
Bacillus
Reaction
pumilus
Bacillus
subtilis
(var.
globigii)
Bacillus
Clostridium
subtilis
sporogenes
8 .0xl0“6
H 2 + discharge
4.3xl0“3
1 .6x10""^
^.lxlO’’3
NgO + discharge
9.3x10”^
-A
3.9x10 *
1 .5x 10
1 .6xlo"'3
•H + NOg •* »0H + NO
1 .9xl0"2
1 .2xl0"3
7 .8x 10*’^
2 .9xlO_2f
*h+o2+h2o -» *ho2+ h 2o
9 8x 10"2
1 ,2xl0”3
2 .9x 10"^
3 «lxlo“3
.
(Data are presented in S/S )
0
..h.
4;_
M
4 ;>
Bo pumilus appears to be the most resistant to free radical
attack.
This is consistent with the resistance of B.
pumilus to ionizing radiation and its use as a biological
indicator for sterilization with gamma radiation.
Without
knowing the concentrations, it is not possible to compare
the absolute sporicidal activity of the radicals generated.
Sporicidal Activity of Water and Microwave Discharge
Simultaneous generation of hydrogen and hydroxyl
radicals was conducted by flowing water vapor through the
discharge cavity for 30 minutes.
The results of this study
on C. sporogenes spores are presented in Figure 9*
The
dissociation of water vapor into radicals by microwave
discharge produced results similar to those for hydrogen
atoms.
The somewhat reduced activity observed with HgO may
be due to the reaction between »H and *0H radicals which
would decrease the amount of °H available for sporicidal
activity.
Alternatively, it might simply reflect that a
somewhat smaller radical concentration is obtained from H 20
compared to that obtained from H 2 dissociation.
Sporicidal Activity of Hydrogen Peroxide
and Microwave Discharge
Sporicidal tests with HgOg/microwave discharge were
conducted by flowing 3 0 $ H 202 through the discharge cavity
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1
room
temp.
1
1x 10
1x10
-2
1x10
5
10
15
20
25
Distance from Discharge Cavity (inches)
Figure 9. Effect of Temperature and Distance
on Sporicidal Activity of Water and
Microwave Discharge with Clostridium
sporogenes Spores on Glass Slides
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for 30 minutes.
The sporicidal activity of 30$ H 202 alone
without the microwave discharge was also studied.
Test
results on B. subtilis spores indicated (figure 10) that
the sporicidal activity of 30$ H 202 with and without the
discharge are about the same.
Apparently, the microwave
discharge did not dissociate all the H 202 flowing through
the cavity.
The 30$ ^ 2 p z a-*-one
sufficient sporicidal
activity to obscure any activity due to radicals generated
by the discharge.
Effect of Carriers on Sporicidal Activity of Radicals
The effect of the carriers on sporicidal activity
was determined by inoculating B. subtilis (var. globigii)
spores on paper discs and glass slides.
A paper disc
and a glass slide were set side by side in an upright posi­
tion 5" away from the discharge cavity.
Results from the
water discharge study (Table 2) indicated that the sporici­
dal activity is better with spores on glass slides than
paper discs.
This is probably due to the porous nature of
the paper discs which allow the spores to penetrate the
disc where they are shielded from the radicals.
It could
also relate to the greater reactivity of the cellulosic
paper substrate when compared to the glass slide.
This
greater reactivity could consume radical species at the
expense of their attack on the spores.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
46
1
1x10
1x10
1x 10
1
-2
4
a s HgOg alone at room temp,
b i E o0 o/discharge at room temp
d s H^O^/discharge at 60°C
5
10
15
20
25
Distance from Discharge Cavity (inches)
Figure 10. Effect of Temperature and Distance on
Sporicidal Activity of 30$ **2°2
and without Microwave Discharge on
Bacillus subtilis Spores
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE
2
EFFECT OF CARRIERS ON SPORICIDAL ACTIVITY OF HgO/MICROWAVE
DISCHARGE WITH BACILLUS SUBTILIS (VAR. GLOBIGII) SPORES AT
0.5 TORR PRESSURE AND 100 WATTS DISCHARGE FOR 30 MINUTES
Initial Number
Carrier
of Spores (SQ )
s/S o
Glass Slides
1 .50xl 05
l.^xlO"2
Paper Discs
1 .50xl 05
2 o60xl 0_1
48
Spores inoculated on glass slides were observed to
clump when examined with a light microscope.
Since radi­
cals will probably react with only the outermost layer of
spores, the inner layers of spores are protected from
radical attack.
This is probably the main reason that
total kill was not observed when spores inoculated on
glass slides were exposed to radicals generated by micro­
wave discharge.
Ability of Radicals to Diffuse Through Porous
Polyethylene (Tyvek) Packaging Material
Sporicidal Tests
These tests were conducted to determined the abi­
lity of various radicals to penetrate Tyvek packaging
and kill B. subtilis (var. globigii) spores on glass
slides.
One unpackaged glass slide and one Tyvek-
packaged glass slide were set side by side in an up­
right position 4" away from the discharge cavity.
The
side containing the spores was placed facing the stream
of radicals.
The results of this study (Table 3) indi­
cated that :
(a) Radicals generated 4 ” away from spores can deacti­
vate unpackaged spores.
(b) None of the radicals evaluated exhibited a signifi-
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TABLE 3
EFFECT OF TYVEK PACKAGE ON SPORICIDAL ACTIVITY
OF RADICALS WITH BACILLUS SUBTILIS (VAR.
GLOBIGII) SPORES ON GLASS SLIDES
Initial
Reaction
Glass Slide Condition
Number of
Spores
Unpackaged
Packaged
vSo'
.
H 2 + discharge
1.93xl06
5»18xl0~^
6 32x 10_1
•H + N02
1.93xl06
9.17x10"^
7.36X10"1
.
4.62X10”4
4.94X10"1
.
*0H + NO
•h+o2+h2o -* »ho2+h2o
1 58x 106
NgO + discharge
1 58x 106
5.19x10“^
5.95X10”1
C02 + discharge
1.25xl06
2.88xl0“3
8.80xl0-1
02 + discharge
1.57xl06
3.95X10”1
2.10X10"1
I.l6xl06
3 36x 10“2
7.93X10"1
N2 + discharge
1.72xl06
9.07xl0"3
5.17xl0_1
Ar + discharge .
1.72xl06
2 67x 10-2
6.40xl0_1
He + discharge
1.72xl06
2.15xl0"2
5.^7xl0-1
02 + discharge
(0.1 torr)
.
.
(Data are presented in S/SQ )
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
cant level of activity against Tyvek-packaged spores.
(c) The low sporicidal activity obtained with Og gas with
microwave discharge is probably due to the rapid reac­
tion of the oxygen atom with a molecule of oxygen to
form ozone, and the low sporicidal activity of ozone
compared to oxygen atom.
The fact that greater spori­
cidal activity was obtained with 0^ gas at 0.1 torr
than 0,5 torr pressure is consistent with this expla­
nation.
Also, at 0.5 torr pressure significant spori­
cidal activity was obtained inside the Tyvek-packaged
spores on glass slides.
This kill was probably due to
ozone which could pass through the porous package.
(d) *H, »0, »0H, and 'HO^ radicals have about the same
sporicidal activity against B. subtilis (var. globigii)
spores under the test conditions.
This is probably
because the radicals kill all of the exposed spores,
but due to clumping, some spores are protected from all
of the radicals.
(e) Nitrogen atom and the metastable Ar and He inert gases
also exhibited some sporicidal activity, but are not
as active as the other species discussed previously.
Wetting Property Tests
Since reduced sporicidal activity was obtained inside
of Tyvek packaging, a test was conducted uo ueoexiume one
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ability of microwave generated radicals to modify surfaces
inside of a Tyvek package.
This test, which measures the
wetting time for silk sutures
80
exposed to various radicals,
provides an independent measure of the ability of radicals
to penetrate packaging and modify surfaces.
Three sets of silk suture loops were located in the
chamber at a distance of 5 "» 15"» and 25" from the microwave
discharge.
Both unpackaged and Tyvek-packaged sutures were
exposed to radicals from the microwave discharge for 30
minutes.
The treated suture loops were then transferred
into a water solution containing 0.05$ Pluronic P104 sur­
factant that was gently stirred with a magnetic stirrer.
The time required for the suture to sink to 'the bottom of
the solution was recorded as the wetting time for the test
sample.
Microwave discharge with Hg, HgO, and 30$H202 were
used in this study.
A control sample exposed to hydrogen
gas at 0.5 torr pressure was used to determine the sinking
time for silk suture loops
radicals.
that were not exposed to any
The results of this study (Table
indicated
th a t :
(a) Radicals can penetrate Tyvek packages and make the
silk suture surface more hydrophilic.
(b) After irradiation, unpackaged silk sutures have shorter
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE
EFFECT OF RADICALS GENERATED BY MICROWAVE DISCHARGE ON WETTING TIME OF
SILK SUTURE LOOPS IN 0.05% ?10b SURFACTANT SOLUTION AFTER DISCHARGING
WITH 100 WATTS POWER AT 0.5 TORR PRESSURE FOR 30 MINUTES
Silk
Reaction
Distance from Discharge Cavity
Suture
Condition
Vacuum Control
5"
Unpackaged
15"
25"
25^ seconds
Unpackaged
immediately
Packaged
^ seconds
Unpackaged
immediately
immediately
11 seconds
17 seconds
immediately
immediately
3 seconds
Packaged
9 seconds
39 seconds
110 seconds
Unpackaged
immediately
6 seconds
16 seconds
seconds
109 seconds
183 seconds
H 2 + Discharge
HgO + Discharge
30$ HpOp + Discharge
Packaged
U9
Lr\
ro
wetting time (are more hydrophilic) than Tyvek-packaged
sutures.
(c) Hydrogen atoms can modify surface at a greater distance,
both inside and outside of a Tyvek package, than
hydroxyl radicals.
The Tyvek packaging used in this study had a maximum
pore size of 10 microns.
In addition, the pores present
in the spunbounded polyethylene structure prevent a tortous path.
Apparently, the reduced activity with Tyvek-
packaged spores is due to interaction of the radicals with
the packaging material.
Radicals can diffuse through
Tyvek packaging but the number of radicals penetrating
the package is not sufficient to provide good sporicidal
activity.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
CONCLUSION AND D ISC U SSIO N OF MICROWAVE DISCHARGE
Microwave discharge studies have demonstrated that
hydrogen, oxygen, hydroxyl, and hydroperoxyl radicals are
sporiciaaiiy active species.
Higher temperature increases
the sporicidal activity of hydrogen and hydroperoxyl radi­
cals hut has significantly less effect on the sporicidal
activity of oxygen and hydroxyl radicals.
These results,
indicate that hydrogen and hydroperoxyl radicals are less
reactive than oxygen and hydroxyl radicals.
The tempera­
ture effect observed with the hydrogen radical is consis­
tent with the increased sporicidal activity attributed
to the hydrogen radical at increased temperature by Friedman and Grecz.
17
Aerobic spores were found to be more resistant to
the radicals than anaerobic spores.
This is consistent
with the fact that aerobic spores that use oxygen for
energy-yielding and biosynthetic reactions have developed
elaborate defense system to deal with the superoxide free
radical, hydroxyl free radical and hydrogen peroxide
formed by the univalent reduction of oxygen.
Apparently,
deactivation can be affected by suppling these radicals
in concentration greater than those cell's enzymatic defen­
ses can handle.
54
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The results of this work show that the distance
from the discharge has a significant effect on the spori­
cidal activity of the radicals.
All the active species
demonstrated a definite loss of activity at long distances
from the point of discharge.
The greatest lost of acti­
vity was obtained with the more reactive radicals, i.e.
hydroxyl and oxygen free radicals.
In addition, the spo­
ricidal activity of the radicals appear to be mainly a
surface effect.
The radicals are ineffective at killing
spores on a reactive porous surface, and do not give total
kill of clumped spores on a smooth surface.
The radicals also demonstrated a limited ability to
penetrate porous polyethylene packaging material with a
maximum pore size of 10 microns.
Radicals can diffuse
through Tyvek packaging but the number of radicals pene­
trating the package is not sufficient to provide good
sporicidal activity.
The results of this work indicate that in order to
achieve good sporicidal activity with packaged articles,
the radicals need to be generated on or near the spore
surface and probably inside the package.
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RESULTS FROM RF DISCHARGE
The power applied to the plasma from the RF generator
can "be read indirectly from the wattmeter.
The wattmeter,
which is located between the RF generator and the plasma
chamber, can read the forward power from the RF generator
and the reflected power from the load.
The net power
applied to the plasma is the difference between the forward
power and the reflected power.
power is less than
In general, the reflected
of the forward power.
Temperature Measurement and Control
The main purpose of this research is to investigate
the interaction of free radicals with spores.
Since high
temperatures can be generated with RF discharges, and high
temperatures by themselves will deactivate spores, conside­
rable time was spend trying to measure and control the tem­
peratures objects experienced when exposed to an RF dis­
charge .
The first attempt to measure the temperature of
objects exposed to plasma was made with temperature indi­
cating crayons (Tempilstik).
Ten small pieces of crayons
with melting points ranging from 51»7°C to 176.7°C, with
56
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
57
13«9°C intervals, were put on a filter paper and placed
horizontally at the center of the RF plasma chamber.
Tyvek
packaging, which melts at about 130°C, was also used to
determined if the temperature indicated by the crayons
corresponded to the melting point of the Tyvek packaging.
^ o C* 4"
j-cJb u
1 +rj
j. c o u x u o
a + a / ^
v u u u u w
i %-»
j_n
« n w
dii
r\l
pj.abiua
f m#>*K1 a
\x a u i c
4
A v>/^
vt^ j
^ A A 4- /A
— ocu
— ■■
j..........
liiuiua
£f A
A
j
that the temperature measured by the crayons was signifi­
cantly lower than the temperature experienced by the Tyvek
packaging when both were exposed to the same plasma.
Appa­
rently, temperature indicating crayons do not indicate the
temperature of objects exposed to RF discharge.
A Fluoroptic thermometer was then used to measure
the temperature of all objects exposed to RF discharge in
this research.
Polyethylene, nylon, and stainless steel
blocks, with dimensions 0.5”x0.5"xl.0", were used with the
Fluoroptic thermometer.
Two holes were drilled in each
block so that the temperature could be monitored at the
surface and the center of each block.
The Fluoroptic ther­
mometer was first used to monitor the temperature of the
nylon block with the probe at the center position.
Besides
continuous plasma, pulsed plasma at the ratio of 1:2 (0.5
ms on and 1.0 ms off) and 1:5 (0.5 ms on and 2.5 ms off)
were also used to study the effect of pulsing the RF power
on the temperature of objects exposed to plasma.
The re­
sults of this study (Figure 11), which was conducted at
2.49 MHz frequency, i.5 torr plasma and 150 watts power,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE 5
EFFECT OF PRESSURE ON TEMPERATURE AND
TYVEK PACKAGE AT 904 KHZ FREQUENCY
AND 200 WATTS PLASMA FOR 5 MINUTES
Temperature
Condition
Pressure
Indicated
of
by Crayon
Tyvek Package
(torr)
(°c)
< 51.7
0.5
did not melt
4
A
J. • V
51=7-65=6
melted
1.5
65.6-79.5
melted
1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
100
continuous
80
60
Temperature
1 s2 pulsed
(°C)
ls5 pulsed
20
10
Exposure Time (minutes)
Figure 11. Temperature of 0.5"x0.5"xl.0" Nylon Block Exposed to RF Plasma
at 2.^9 MHz Frequency,
1.5 Torr Pressure and 150 Watts Power
(probe was located at the center of the block)
\J\
\o
60
indicated that the temperature of the nylon block increases
with increasing plasma exposure time and decreases with
increasing the off time with pulsed plasma.
Tyvek packa­
ging melted in less than 5 minutes with continuous plasma,
but was not damaged in either 1:2 or 1:5 pulsed plasma.
Apparently, pulsing the plasma power will effectively con­
trol the temperature of objects exposed to the plasma.
The
difference of temperature between the Tyvek packaging and
the nylon block will be explained later.
Since continuous
plasma melted the Tyvek packaging and 1:5 pulsed plasma
required a longer time to achieve sterilization, 1:2 pulsed
plasma was used in all subsequent studies unless noted
otherwise.
The heating effects of a 1:2 pulsed RF plasma at 150
watts power on the nylon, polyethylene, and stainless steel
blocks are presented in Figure 12.
The results indicated
that the temperature of the metal block was slightly lower
than the temperature of nonmetal blocks and with each block
the surface and bulk temperatures of the materials were
approximately the same.
This illustrates that metals are
not preferentially heated under the test condition and that
a significant temperature differential does not exist in
materials exposed to low temperature plasma at 2 A 9 MHz
frequency.
Additional temperature studies with the nylon
block showed that the temperature of the block increased
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Surface Temperature
70 ■
60--
60 ••
Temp,
40
Temp ■
<°C)
(°C)
p s polyethylene
20
to 3 tl
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Bnlk Temperature
4030-
20
.
n s nylon
10
--
s : stainless Steel
10 .1
3
5
7
9
11
13
15
Exposure Time (min.)
Figure 12. Temperature of 0 .5"x0,5"xl.0" Stainless Steel, Polyethylene,
and Nylon Blocks Exposed to RF Plasma at 2.49 MHz Frequency,
1,5 Torr Pressure and 150 Watts of 1:2 Pulsed Plasma
CN
I-*
with increasing plasma pressure at constant power (Figure
13) and plasma power at constant pressure (Figure 14).
The temperature recorded by the probe alone,' without
being inserting into any block, located at the center of
plasma chamber was also studied.
The results (Figure 15)
show that the temperature recorded was significantly higher
than the temperature obtained when the probe is inserted in
the blocks. To try and understand this difference, the tem­
perature of nylon blocks with the dimensions 0.25"x0.5"xl.0"
and 0.125"x0.5"xl.0" (i.e., higher surface area to volume
ratio) were determined under the same plasma exposure
conditions.
Only one hole was drilled at the center of
these smaller blocks.
The results of this experiment
(Figure 16) indicated that the temperature of an object ex­
posed to low temperature plasma increases as the ratio of
surface area to volume increases.
Apparently, the tempera­
ture recorded by the probe was the temperature of the Tef­
lon coating on the probe.
The surface area to volume ratio
of the Teflon coating around the phosphor is about 178
inch
.
The high probe temperature is, therefore, consis­
tent with the high surface area to volume ratio.
This
illustrates that the temperature an object reaches when
exposed to plasma will depend on the size, shape and, pre­
sumably, the heat capacity of the object.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
65
2.0 torr
60
55
1.5 torr
50
Temperature
1.0 torr
(°C)
0.5 torr
35
30
25
20
1
2
6
7
8
9
10
11
12
13
14
Exposure Time (minutes)
Figure 13. Effect of Pressure on Temperature of Nylon Block Exposed to
HgO Plasma at 2.49 MHz Frequency and 150 Watts of Is 2 Pulsed
Plasma
Ox
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
200 watts
150 watts
Temperature
(°C)
40
100 watts
50 watts
20
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Exposure Time (minutes)
Figure 14. Effect of Power on Temperature of Nylon Block Exposed to Air
Plasma at 2.49 MHz Frequency,
1.5 Torr Pressure and 1:2 Pulsed
Plasma
On
-p-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
110
probe alone - probe is in the
100
center of the
plasma chamber
80
Temperature
70
(°C)
block ~ probe is in the
center of the
block
20
Exposure Time (minutes)
Figure 15. Temperature of Fluoroptic Thermometer Probe and 0.5"x0.5"xl.0"
Nylon Block at 1.5 Torr Pressure, 2.^9 MHz Frequency and 150
Os
Watts of 1 12 Pulsed Plasma
''A
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
110
probe
100
80
Temperature
(°C)
60
20
Exposure Time (minutes)
Figure 16. Temperature of Fluoroptic Thermometer Probe and Nylon Blocks
at 2.^-9 MHz, 1.5 Torr Pressure and 150 Watts of ls2 Pulsed Plasma
On
ON
Tyvek-packaged samples of polyethylene microfibers
(PEMF), with approximately 3*0x10
inch-
surface to volume
ratio, were exposed to 15 minutes of air plasma with 150
watts of 1j2 pulsed power at 1.5 torr pressure and 3,89 MHz
frequency.
The untreated PEMF showed a thermal history at
about 56°C (Figure 17) which was probably the previous
processing or storage temperature of PEMF samples.
The
treated PEMF showed a thermal history at about 85°C (Figure
17) which was apparently the highest temperature the PEMF
reached in the plasma.
Since the thermal history of
treated PEMF was higher than the thermal history of un­
treated PEMF, the 56°C thermal history was wiped out and
only the 85°C thermal history showed up in the scan of the
treated PEMF.
The temperature reached by the Fluoroptic
thermometer probe under the same reaction condition was
approximately 80°C.
These results indicated that the tem­
perature reached by the Fluoroptic thermometer probe is
close to the temperature experienced by small items with
large surface to volume ratio in RF plasma under the test
conditions.
The results of the probe temperature exposed to air
at 150 watts of continuous plasma are .presented in Figure
18.
Unlike the pulsed plasma, where the probe temperature
after an initially rapid increase continued to increase
slowly with increasing exposure time, the probe temperature
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Heat
Flow Rate
(joule/sec.)
control:
untreated
PEMF
plasma
treated
PEMF
27
77
127
Temperature (°C)
Figure 17. Heat History from Differential Scanning
Calorimeter of polyethylene microfibers (PEMF)
Samples Treated with 15 Minutes of Air Plasma
at 150 Watts of 1:2 Pulsed Power and 1.5 Torr
Pressure
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
180
160
continuous
120
Temperature
100
1 s2 pulsed
(°C)
80
60
20
Exposure Time (minutes)
Figure 18. Temperature o.f Fluoroptic Thermometer Probe in Air Plasma
at 1.5 Torr Pressure, 2.^9 MHz Frequency and 150 Watts Power
O'.
VO
exposed to continuous plasma reaches a steady state after
a few minutes.
This should be the translational (gas)
temperature of the plasma.
Prom the results of the probe
temperature with continuous and pulsed plasma, it is easy
to see why the Tyvek packaging was melted in 5 minutes of
continuous plasma at 150 watts, but not in 15 minutes of
i :2 pulsed plasma.
Due to the high surface area to volume ratio of the
Teflon coating on the probe, the temperature readout from
the probe alone was used as an upper limit on the tempera­
tures that nonmetallic objects will reach in the RP plasma.
The probe temperature can also be used as an upper tempe­
rature limit for metallic articles as long as they are not
preferentially heated by radio frequencies used to generate
the plasma.
Effect of Frequency on Temperature
The effect of radio frequency on temperature was
evaluated by measuring the temperature of the Fluoroptic
thermometer probe and 0 .5 "x0 .5 "xl.0" polyethylene, nylon,
and stainless steel blocks exposed to 150 watts of Is2
pulsed plasma at 1,5 torr pressure for 15 minutes.
frequency range examined was 598 KHz to 3.89 MHz.
The
To eva­
luate this broad frequency range, three RF coil were used.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Since f«l/JlC and ]>N^A/l, £where f is the frequency, L is
the inductance of the coil, C is the capacitance of the
capacitors, N is the number of turns of the coil, A is the
area under the coil and 1 is the length of the coil/] for a
given set of variable
air capacitors each coil has a spe­
cific frequency range over which it will operate.
The
physical properties of the three coils used in this study
are presented in Table 6.
The major difference of these
three coils is the number of turns.
The number of turns
for the three coils were chosen so that some frequency
overlap occurred between coils.
The results of this study are presented in Figure 19.
The results indicate that temperature increases with in­
creasing frequency for a given coil.
Also, at a given
frequency, temperature increased as the number of turns in
the coil increased.
This can be explained by the equation
v(t)=L(d(i)/d(t)), where v(t) is the induced voltage across
the coil- L is the inductance of the coil, and d(i)/d(t)
corresponds to the frequency.
In a plasma, the induced
voltage accelerates the electrons and these electrons
transfer their energy to the molecules by collisions.
An
increase in induced voltage thus raises the average elec­
tron energy.
The voltage induced, and therefore the tem-
erature of the probe and objects exposed to the plasma
should increase with increasing frequency and inductance.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
72
TABLE 6
PHYSICAL PROPERTIES OF RF COILS USED
TO STUDY THE EFFECT OF FREQUENCY ON
TEMPERATURE AND SPORICIDAL ACTIVITY
Coil A
Coil B
Coil C
0.D.=0.25"
0.D.=0.25"
0.D.=0.25"
Copper Tubing
1.D.=0.125”
1.D.=0.125"
1.D.=0.125"
Frequency
560 KHz
1.02 MHz
1.77 MHz
to
to
to
1*31 MHz
2*54 MH z
4.20 MHz
22
12
9.5”
9.5"
9.5"
11.5"
10"
10"
Size of
Range
Number
of Turns
6
Diameter
of Coil
Length
of Coil
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
HO-
100
PE
-
probe
stainless steel
polyethylene
nylon
90-
80
--
Temp
70-
(°C)
60
■-
50PE
PE
20
-
0.6
8
2.2
2.6
Frequency (MHz)
Figure 19. Effect of Frequency on Temperatures of Fluoroptic Thermometer Probe
and 0 .5"x0.5"xl.0" Blocks after 15 Minutes of 150 Watts of Is2
Pulsed Plasma at 1.5 Torr Pressure
V.0
As with previous temperature measurements (all the
temperature measurements discussed previously at 2.^9 MHz
were conducted with the 12 turn coil), the prohe temperature
was significantly higher than the temperature of metal and
nonmetal blocks exposed to the plasma.
The temperature of
metals and nonmetals were approximately the same except at
frequencies below 1.0 MHz where significantly higher tempe­
ratures were obtained with the metal block.
bly due
quency.
This is proba­
to direct RF heating of the metals at the lower fre­
That direct RF heating occurs at 2 A 9 MHz (6 turn
coil) with metal objects was determined by exposing the
stainless steel block to 100 watts of continuous RF energy
at atmospheric pressure where no plasma was generated.
The
results (Figure 20) indicated that the temperature of the
0.5"x0.5"xl.0" nylon block and Fluoroptic thermometer probe
alone remain the same throughout the 10 minutes exposure
time, but the temperature of the 0.5"x0.5"xl.0" stainless
steel block increased with increasing exposure time.
Appa­
rently, at low frequencies the RF energy used to generate
the plasma has a direct heating effect on metal objects.
Sporicidal Activity of N^O. 0»» H^. H^O and H ^O^ with
RF Discharge
at 2.49 MHz with 12 Turn Coil
B. subtilis (var. blobigii) spores were used to study
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
stainless steel
35
Temperature
(°C)
30
2
■ :
nylon and probe alone
25
20
10
Exposure Time (minutes)
Figure 20. Effect of Radio Frequency on Temperatures of Fluoroptic
Thermometer Probe and 0 .5"x0.5"xl.0" Nylon and Stainless
Steel Blocks at 2.^9 MHz (6 Turn Coil), atmospheric
Pressure and 100 Watts of Continuous Plasma
the sporicidal activity of various chemicals in an RF dis­
charge.
This organism was chosen because of its reported
high resistance to hydrogen peroxide
81
and its resistance
to radicals observed in the microwave discharge study.
As
in the case of the microwave discharge studies, the spori­
cidal activity of the RF discharge is expressed as S/SQ .
The chemicals chosen were those, which according to litera­
ture references, should produce »H, *0, .OH, or ‘HOg*
Microwave discharge studies indicated that in order
to achieve good sporicidal activity, the active species
must be generated on or near the spore surface inside
the Tyvek package.
Therefore, a "pretreatment time" was
used to allow the chemical to contact the article prior to
the application of the RF energy.
Tyvek-packaged B. subtilis (var. globigii) spores on
paper discs were first placed in the center of the plasma
chamber, the chamber was closed and a vacuum was drawn to
a pressure below 0.05 torr.
An aqueous solution or gas
was then introduced into the chamber and the pressure in
the chamber maintained at 1.5 torr with a throttle valve
controller.
The gas or vapor remained in the chamber for
10 minutes before 150 watts of Is2 pulsed plasma was gene­
rated at 2.49 MHz frequency and 1.5 torr pressure for 15
minutes.
After the gas or vapor pretreatment and plasma
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
77
treatment cycle, the vacuum was released and the paper
discs were assayed for survivors.
For NgO, 02 and H 2 gases, the gas was slowly flowed
into the chamber during the 10 minutes pretreatment time
and ceased before the generating of plasma.
For H 202 , two
portions of 1.25 mg (0.025 ml of 5/0 of H 2Q2 were injected
at zero and 5 minute time points.
For HgO, two portions
of 0.025 ml of HgO were injected at zero and 5 minute time
points.
The sporicidal test results (Table 7) indicated that
only the combination of H 202 and plasma deactivated all the
spores.
No appreciable sporicidal activity was obtained
with H 20/plasma and H 2/plasma.
The reduced activity with
H 20/plasma and Hg/plasma may be due to s
(1) The high bond
dissociation energy of H 20 and H 2 compared to HgOg. (2) The
H 202/plasma generated some active species that H 20/plasma
and H 2/plasma do not possess, or (3) A synergism exists
between H 202 and the radicals generated by the H 202/plasma.
The N20/plasma had better sporicidal activity than the
02/plasma.
This is consistent with the results of the
microwave work and may relate to the lower bond dissocia­
tion energy of N?0 compared to Og.
Although the bond
dissociation energy of N20 to generate 0 atom is lower
than the bond dissociation energy of
to generate *0H
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78
TABLE 7
SPORICIDAL ACTIVITY OF PLASMA WITH 0 ^
NgO, HgO,
h 2o2 and h 2 ON TYVEK-PACKAGED B. SUBTILIS
(VAR. GLOBIGII) SPORES ON PAPER DISCS
Gas
Initial
Sporicidal
Number of
Activity
Spores
(s/s0)
(So>
H2°2
h
2o
H2
n 2o
°2
3.4x 105
0
ZAxlO5
IcO
l.lxlO5
7.7xlO_1
1.6xl05
3.1xl0_1
1.3xl06
7.2x10"*11
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
radical, the sporicidal activity from NgO/plasma does not
compare to the total deactivation obtained with HgOg/plasma.
The sporicidal activity of HgO/plasma system was
further evaluated using the same pretreatment process but
with higher powers.
Since Tvvek package melted at higher
powers of 1:2 pulsed plasma, 1:5 pulsed plasma with 30 mi­
nutes plasma treatment time was used.
The results of this
study (Table 8) indicated that sporicidal activity remains
about the same with increasing power levels and there is
no apparent synergism between water vapor and plasma for
deactivaing spores.
Effect of Parameters on S-poricidal Activity of
Hydrogen Peroxide/Plasma
Since H^Og/plasma was the only RF discharge system eva­
luated that exhibited good sporicidal activity, the effect
of the various operating parameters in the HgOg/plasma sys­
tem on sporicidal activity were investigated in detail.
Effect of
Pretreatment Time
The effect of pretreatment time with hydrogen peroxide
vapor on sporicidal activity of hydrogen peroxide/plasma was
determined by pretreating samples for 1, 2, 3, 4, 5» and 10
minutes intervals with 1.25 mg (0.025 ml of 5%) of hydrogen
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80
TABLE 8
EFFECT OF POWER ON SPORICIDAL ACTIVITY OF HgO/PLASMA
WITH TYVEK-PACKAGED BACILLUS SUBTILIS (VAR.
GLOBIGII) SPORES ON PAPER DISCS
Power
(watts)
Initial Number
Sporicidal Activity (S/SQ)
of Spores (SQ)
Plasma Alone
HgO/Plasma
200
5.8x10^
1.0
1.0
300
5.8x104
1.0
1.0
400
5.8xl04
500
5.8x10
600
4.6x10^
700
5.2x10
800
2.2X10-5
7.7X10"1
1.6xl0-1
900
1.2X10-5
3 . 6 x 1 0 -1
7 . 8 x 1 0 _1
4
4
5.0X10-1
7.2xl0_1
6.0X10"1
1.0
9.8xl0-1
8.5X10"1
1.0
7.3xlO_1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
peroxide injected at the zero time point.
The samples were
treated with 150 watts of 1:2 pulsed plasma at 1.5 torr
pressure for 15 minutes.
The results of this study (Figure
21) showed that the sporicidal activity increased with in­
creasing pretreatment time up to approximately 5 minutes
where it leveled off.
Therefore, two portions of 1.25 mg
(0.025 ml of 5%) of hydrogen peroxide were injected at zero
and 5 minute time points for the subsequent 10 minutes
hydrogen peroxide pretreatment tests.
Effect of H ^Oo Concentration
The effect of hydrogen peroxide concentration on the
sporicidal activity of the hydrogen peroxide and plasma
system was determined by injecting two portions of 0.025 ml
hydrogen peroxide at zero and 5 minute time points, respec­
tively, thus providing a total pretreatment time of 10 minu­
tes at 1 torr total pressure.
The treated samples were then
exposed to 200 watts of 1:2 pulsed plasma for 15 minutes.
The amount of hydrogen peroxide used for the tests were
1.5 mg (2x0.025 ml of 3$), 2.5 mg (2x0.025 ml of 5%), 5*0
mg (2x0.025 ml of 10$), and 7.5 mg (2x0.025 ml of 15$). A
water/plasma control consisting of a 10-minute water pre­
treatment and a 15-minute plasma treatment, and a 25-minute
hydrogen peroxide control were also conducted.
The results
of this study (Figure 22) showed that (i) No significant
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
82
1 f
plasma
alone
1x10
1x10
-1 ■■
alone
-2
S/S,
IxlO-3 4-
1x10
-k
1x10 J -
2
4
6
8
— 4—
10
Pretreatment Time (minutes)
Figure 21. Effect of H 202 Pretreatment Time on
Sporicidal Activity of HgOg/Plasma with
Tyvek-Packaged Bacillus subtilis (var.
glohigii) Spores on Paper Discs at 1.5
torr and 150 watts of Is2 Pulsed Plasma
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
83
1
alone
1x10
1x10
1
4
total kill
of 2.4x 105
^
1.5
2.5
5.0
spores
7.5
Amount of HgOg Injected (mg)
Figure 22. Effect of HgOg Concentration on Sporicidal
Activity of HgOg/Plasma with Tyvek-Packaged
Bacillus subtilis (var. globigii) Spores
on Paper Discs at 200 watts of 1:2 Pulsed
Plasma
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
sporicidal activity was obtained with water/plasma treat­
ment or hydrogen peroxide treatment alone when the amount
of hydrogen peroxide used was less than 5.0 mg.
(ii) The
sporicidal activity of hydrogen peroxide/plasma increased
with increasing hydrogen peroxide injected,
(iii) The
combination of hydrogen peroxide and plasma was synergistic
at all hydrogen peroxide levels evaluated,
(iv) A strong
synergism was observed when 5*0 mg of hydrogen peroxide was
used with the RF discharge in that total spore inactivation
was achieved,
(v) Total spore inactivation was also ob­
served at 7.5 mg H 2°2'
'*'he s^ronS synergism obtained with
the combination of
and plasma may be due to radical
formation with HgOg in the RF discharge.
Effect of Pressure
The effect of pressure on the sporicidal activity of
hydrogen peroxide/plasma was obtained using 2.5 mg (2x0.025
ml of 5%) of hydrogen peroxide during the 10 minutes pre­
treatment time.
This was followed by 15 minutes of 200
watts of 1:2 pulsed plasma.
The activity was determined
at pressures of 0.5, 1.0, 1 .5 , and 2.0 torr.
The activity
of 15 minutes of air plasma and 25 minutes of hydrogen
peroxide alone were also determined.
The results of this
study (Figure 23) showed that a low level of activity was
obtained with either plasma alone or hydrogen peroxide
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H o0~ alone
Plasma
alone
1x10
1x10
-2
1x10
1x10
1x10
total kill
of 3.j^xlO-5
spores .
0.5
1.0
1.5
2.0
Pressure (torr)
Figure 23. Effect of Pressure on Sporicidal Activity
of H 202/Plasma with Tyvek-Packaged
Bacillus subtilis (var, glohigii) Spores
on Paper Discs at 200 watts of 1:2 Pulsed
Plasma. 2.5 mg of H 202 was used.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
86
alone at all pressures.
The optimum sporicidal activity
with the hydrogen peroxide/plasma system was obtained at
1.5 torr pressure.
The decreased activity at 2.0 torr was
probably due to the reduced average mean free path of the
electrons at higher pressure.
As a consequence electrons
pick up less energy between collisions and shift the elec­
tron energy distribution to lower values.
In addition,
the mean free path of free radicals generated by the elec­
trons would also be reduced.
Effect of Plasma Power
The effect of plasma power on sporicidal activity was
determined using 2.5 mg (2x0.025 ml of 5%) of hydrogen per­
oxide at 1.5 torr pressure and plasma power levels of 5 0,
100, 150, and 200 watts.
As before, the samples were first
treated for 10 minutes with hydrogen peroxide and then ex­
posed to 1:2 pulsed plasma for 15 minutes.
Control tests
consisting of 15 minutes plasma alone and 25 minutes hydro­
gen peroxide alone were also conducted.
presented in Figure 24.
The results are
A low level of sporicidal activity
was obtained with air plasma alone at all power loads eva­
luated and hydrogen peroxide and plasma at 50 watts.
Sig­
nificant sporicidal activity was obtained with the hydrogen
peroxide plus plasma system at 100 watts power, and total
deactivation was achieved at 150 and 200 watts.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
87
plasma 'alone
1x10
1x10
-2
1x10
1x10
total kill
of 1.8x10^
spores
50
100
150
200
Power (watts)
Figure 24. Effect of RF Power on Sporicidal Activity
of H 202/Plasma with Tyvek-Packaged
Bacillus subtilis (var. globigii) Spores
on Paper Discs at 1.5 torr Pressure.
2.5 mg of H 202 was used.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Depth of Penetration of Hydrogen Peroxide and RF Discharge
System with Paper Disc Substrate
All the test results discussed previously in RF dis­
charge work used the plate count method to assay for sur­
vivors,
Each paper disc was vortexed in stripping fluid
with catalase for one minute after the test to recover
surviving organisms.
With one minute vortexing only the
outer, layers of the paper discs were macerated.
A test
conducted with a two minute vortex time, which completely
macerated the disc, was conducted at conditions that indi­
cated sterility "by the one minute vortex method, i.e., 10
minutes pretreatment with 2.5 mg (2x0.025 ml of 5%) of
hydrogen peroxide followed by 15 minutes of 150 watts of
1:2 pulsed plasma.
Control experiments with 15 minutes of
plasma alone and 25 minutes of hydrogen peroxide alone were
also conducted.
The results of these experiments (Table
9) show that m o r e ■surviving organisms were obtained after
treatment with plasma alone, hydrogen peroxide alone, and
hydrogen peroxide/plasma with the two minute vortex reco­
very method.
Apparently, spores were present deep in the
center of the paper disc that were not deactivated by the
hydrogen peroxide process and were not recovered with the
one minute vortex method.
As a result of this work the
condition required to achieve total kill, based on the
two minute vortex process, had to be redetermined,
A se-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
RQ
w/
TABLE
9
EFFECT OF VORTEX TIME ON SPORICIDAL ACTIVITY OF
h
2 o 2/p l a s m a WITH TYVEK-PACKAGED B. SUBTILIS
(VAR. GLOBIGII) SPORES ON PAPER DISCS
--------------------
1 minute
2 minutes
Vortex
Vortex
2.2xl05
3.0xl05
Plasma Alone
2.teL0“1
9C3X10"1
HgOg Alone
4.0xl0_1
8.0xi0_1
0
6.6xl0-^
Initial Number
of Spores (SQ )
HgOg/Plasma
i
1
!
Sporicidal Activities are presented in S/SQ
Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission.
90
ries of experiments were conducted to determine the effects
of hydrogen peroxide pretreatment time and hydrogen peroxide
concentration on the sporicidal activity of the hydrogen
peroxide/plasma system with the two minute vortex recovery
method on Tyvek-paekaged B. suhtilis (var. globigii) spores
on paper discs at 1.5 torr pressure and 2.4-9 MHz frequency.
These effects were determined "by pretreating samples for
5, 10, 20, and 30 minutes intervals with 1=25 mg (0=025 ml
of 5$)» 2.5 mg (0.025 ml of 10$), and 3*75 mg (0.025 ml of
15$) of hydrogen peroxide injected at the zero time point
and then exposing the treated samples to 15 minutes of 150
watts of 1:2 pulsed plasma.
The results of these experiments (Figure 25) show that
the sporicidal activity increased with increasing hydrogen
peroxide concentration and with increasing hydrogen per­
oxide pretreatment time.
Unlike the results obtained with
the one minute vortex method, where the sporicidal activity
levels off after a 5 minute pretreatment period, signifi­
cantly better sporicidal activity was obtained with a 10
minute pretreatment with the two minute vortex method.
Apparently, a longer pretreatment time is required for the
hydrogen peroxide to diffuse to the spores deep in the
center of the paper disc.
All subsequent tests were there­
fore conducted with a 10 minute pretreatment time, followed
by 15 minutes of 150 watts of 1:2 pulsed plasma.
Hydrogen
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
91
2
1.25 mg
3
1x10 -1
2.5 mg
5
1x10
\ 3.75 mg
6
S/S,
1x10 -3 ..
1x10
7
_Zj.
2.5 mg
1 : plasma alone
1x10 -5
2-4
H 202 alone
5-7
H202/plasma
5
10
20
30
Pretreatment Time (min.)
Figure 25* Effect of HgOg Concentration and Pretreatment
Time on Sporicidal Activity of H 202/Plasma
with Tyvek-Packaged Bacillus subtilis (var.
globigii) Spores on Paper Discs as Obtained
Using the 2 Minute Vortex Analysis Method
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
peroxide with 2.5 mg (0.025 ml of 10$) was used for the
test to keep the initial hydrogen peroxide concentrations
to a minimum and to maximize the synergism obtained with
the hydrogen peroxide plasma system.
Effect of Frequency on SDoriCj-daf Activity of Hydrogen
Peroxide and RF Discharge
The effect of frequency on the sporicidal activity
of hydrogen peroxide/plasma was determined with Tyvekpackaged B. subtilis (var. globigii) spores on paper discs
over the frequency range of 1.09 MHz to 3-89 MHz.
Tests
were conducted with a 10-minute pretreatment period with
2.5 mg (0.025 ml of 10$) of hydrogen peroxide and a 15minute plasma treatment with 150 watts of 1:2 pulsed plasma
at 1.5 torr pressure.
sented in Figure 2 6 .
The results of this study are pre­
The sporicidal activity increased with
increasing frequency for a given coil, and, at a given fre­
quency, was highest for the coil with the largest number of
turns.
A comparison of temperature and sporicidal activity
for the two coils is shown in Figure 27.
One interesting
observation is that better sporicidal activity can be ob­
tained at a lower temperature with the 6 turn coil.
Either
total kill or near total kill was obtained at the frequency
range of 3.^9 MHz to 3.89 MHz.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1x10
1x10
o
6 turn coil
1x10
12 turn coil
Frequency (MHz)
Figure 26. Effect of Frequency with 12 Turn and 6 Turn
RF Coils on Sporicidal Activity of HgOg and
Plasma with Tyvek-Packaged Bacillus subtilis
(var, globigii) Spores on Paper Discs
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1120
•110
1x10 -1
••100
1x10
-2
.. 90
•• 80
1x10
S/S0
•70
1x10 - U
•
Temperature
(°C )
-60
1x10
1x10
1
2
3
^Frequency (MHz)
Figure
27. Effect of Frequency on Sporicidal Activity of HgOg/plasma with
Tyvek-packaged B. subtilis (var. globigii) Spores on Paper Discs
and the Temperature of the Fluoroptic Thermometer Probe.
Dashed
lines (curves A & B) represent temperatures obtained with 12 and
6 turn coils, respectively.
Curves C and D represent sporicidal
activity for 12 and 6 turn coils, respectively.
vo
Since the RF voltage induced into the plasma is depen­
dent upon the coil used, one cannot directly compare the
effect of frequency on sporicidal activity over the total
frequency range examined.
However, if one assumes that the
plasma temperature is a measurement of the induced voltage
then a plot of sporicidal activity against frequency at a
constant temperature should illustrate the effect of fre­
quency on sporicidal activity.
Such a plot, Figure 28,
shows that sporicidal activity increases with increasing
frequency at a constant temperature.
Perhaps more signifi­
cantly, this plot suggests that by working at higher fre­
quencies, better sporicidal activity can be obtained at a
lower temperature.
Dissociation of Hydrogen Peroxide with RF Discharge
An experiment was conducted to determine the ability
of various RF power levels to dissociate hydrogen peroxide
in the vapor phase.
Tests were run by evacuating the cham­
ber to about 0.03 torr, switching the pressure control
setting to 1.5 torr (pressure still remained at 0.03 torr),
generating the plasma with 50, 75* 100, 125» and 150 watts
RF power at 3-89 MHz, injecting 2.5 mg (0.025 ml of 10%)
of HgOg into the chamber immediately (pressure increased
to 1.5 torr after injection), changing the RF power to 150
watts after 10 minutes, and exposing the samples to 150
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1x10 -1
1x10
1x10
-k
1x10
1x10
+
Frequency (MHz)
Figure 28. Effect of Frequency on Sporicidal Activity of HgOg/plasma with
Tyvek-packaged B. subtilis (var. globigii) Spores on Paper Discs
at a Constant Temperature.
Temperature were obtained by Fluoroptic
thermometer measurements on a 0 .5"x0,5"xl.0" nylon block.
so
Os
watts plasma for 15 minutes.
All plasma power was pulsed
in a cycle of 0.5 ms on and 1.0 ms off.
The results (Figure
29) of this study, which were conducted on Tyvek-packaged B„
subtilis (var. globigii) spores on paper discs, showed that
significant sporicidal activity was obtained at 50 and 75
watts of power but that all sporicidal activity was lost
when 100 watts of power or higher were applied when hydro­
gen peroxide was introduced after the formation of plasma.
Apparently, at higher power levels most of the hydrogen
peroxide is decomposed before it can diffuse to the site
of the spores.
At lower power levels, sufficient hydrogen
peroxide can diffuse onto the spore surface to provide
significant sporicidal activity with plasma.
The power
required to dissociate hydrogen peroxide is apparently
between 75 and 100 watts of Is2 pulsed plasma.
These
results are consistent with the results found with the
microwave discharge which indicated that the active
species needs to be generated on the spore surface or
inside the Tyvek package in order to have good sporicidal
activity.
Snoricidal Activity of H oO^/Plasma on
Four Bacterial Spores
The ability of the hydrogen peroxide and plasma sys­
tem to deactivate both anaerobic and aerobic spores was
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1x10
1x10
50
75
100
125
150
Power (watts)
Figure 29. Effect of Plasma Power During H 202
Pretreatment on Sporicidal Activity of
H 202/Plasma with Tyvek-Packaged Bacillus
subtilis (var. globigii) Spores on Paper
Discs at 1.5 torr Pressure
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
determined using the same spores as evaluated in the micro­
wave studies.
In the RF discharge study all organisms were
exposed to a process that consisted of : a vacuum of 0.03
torr for 15 minutes, H 202 pretreatment at 3-0 torr with
2.5 mg (0.05 ml of 10$) of hydrogen peroxide for 15 minutes,
and 150 watts of 1:2 pulsed plasma at 1.5 torr pressure and
3.89 MHz frequency for 15 minutes.
All spores were inocu­
lated on paper discs and heat-sealed in Tyvek packages.
The ATCC number and natural resistance of these organisms
can be found in the microbiological methods section of the
introduction.
Control experiments were conducted in which
the sporicidal activity of vacuum alone, hydrogen peroxide
alone, and air plasma alone were also determined.
The
results of this work (Table 10) showed that (1) H 202 alone
killed all the anaerobic C. soorogenes spores.
also the least resistant to plasma treatment.
It was
Unlike the
aerobic spores, anaerobic spores lives in the absence of
oxygen and thus have not developed any defense systems to
deal with various oxidizing agents.
(2) B. numilus spores
were the most resistant spores to the plasma alone.
This
is consistent with the resistance of B. numilus spores to
radiation.
A significant number of B. numilus spores were
deactivated by H 202 vapor.
This demonstrates that spores
which are highly resistant to one process are not necessa­
rily insensitive to another process that involves another
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S P O R IC ID A L A C T I V I T Y
A N A E R O B IC
Spores
of
h
2 o 2/ p l a s m a
SPORES ON P APER D IS C S
So
SYSTEM W IT H A E R O B IC AND
IN S ID E
TYVEK PACKAGE
Vacuum
Plasma
H 2°2
Alone
Alone
alone
0
HgOg/Plasma
0
C . soorogenes
2„2xl05
6.9xlO_1
l.lxlO-2
B. subtilis
1.5xl06
8.9xlO_1
5.9xlO-1
6.8X.10"1
0
2.3xl06
7.8X10-1
7.^-xlO-1
5.7X10"1
0
l.OxlO6
9.6xlO_1
9.5xlO_1
8.5x10“^
0
B. subtilis
(var. globigii)
B . numilus
100
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TABLE 1 0
101
mechanism of action.
(3) B. subtilis (var. globigii) and
B. subtilis spores were the two most resistant spores to
both H 202 and H 202/plasma, however, the combination of H 202
and plasma provides total kill with both organisms.
Compareson between H 202/Piasma and H 202/Heat
A comparison of the ability of H 202/plasma and
H 202/heat to inactivate spores was conducted to try and
better understand the mechanism of kill in the H 202/plasma
system.
In addition, the ability of these processes to
remove H 202 residuals from the carrier paper discs was
determined.
The amount of hydrogen peroxide on the paper discs
before and after the plasma or heat treatment was deter­
mined by spectrophotometrically (Beckmam DU-7 spectrophoto­
meter) measuring the I2 generated at 350 nm By the reac­
tion :
H 202 + 2KI + HgSO^ ----- > I2 + KgSO^ + 2H20
Tyvek-packaged paper discs were first treated with
2.5 mg (0.05 ml of 10$) of H 202 at 3.0 torr for 15 minutes
then followed with either 150 watts of 1:2 pulsed plasma
at 1.5 torr pressure and 3*89 MHz frequency or infrared
heat at 1.5 torr for 5» 10, and 15 minutes.
A 250 watts
Sylvania Infrared lamp was used to heat the H 202 system at
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
102
about the same rate of temperature increase as obtained
with RF plasma.
The temperature was monitored with the
Fluoroptic thermometer probe.
Comparisons of the sporicidal activity obtained with
Tyvek-packaged B. subtilis (var, globigii) spores, and the
level of
Table 11.
c. c.
residual on naner discs are -presented in
- -
Total kill was obtained with 5 minutes plasma
compared to almost insignificant kill with 5 minutes of
heat, even though the temperature
of both system were
approximately the same.
minutes exposure toheat
After 15
an increase in sporicidal activity was obtained but no
hydrogen peroxide residuals were removed from the paper
discs.
In contrast, 15 minutes of plasma
removed about 95%>
of HgOg on the paper discs.
Apparently, the H 202/plasma process is not the same as
the H 202/heat process.
The H 202/plasma system both deacti­
vates spores and removes H 202 from substrates during the
plasma treatment.
Since H 202 was not completely dissoci­
ated from paper discs after plasma treatment, and since heat
will enhance the sporicidal activity of H 2C>2 , a portion of
the HgOg/plasma sporicidal activity may be due to H 202 and
heat.
Temperature will, therefore, enhance the sporicidal
activity of H 202/plasma process.
A separation of the effect
of heat on the sporicidal activity of H 2C>2 as compared to
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EFFECT OF PLASMA AND HEAT EXPOSURE TIME ON SPORICIDAL ACTIVITY, H 202
RESIDUALS, AND TEMPERATURE INCREASED OF HgOg/PLASMA AND HgOg/HEAT
Exposure
15 min. H 20 2 Pretreatment
Plasma
in
H 2°2
or Heat
(minutes)
0
15 min. H 202 Pretreatment
+ Plasma
Time of
o
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TABLE 11
Residual
(microgram)
7-lxlO"1
381
+ Heat
Temperature
Increased
s/ So
(°c)
0
H 2°2
Temperature
Residual
Increased
(microgram)
(°C)
7 .lxlO-1
381
0
5
0
157
43.9
8.4X10-1
579
41 .2
10
0
71
53.2
2.6X10"1
608
49.7
15
0
20
56.4
6.3x10
568
54.5
-k
o
104
radicals generated from the decomposition of the HgOg would
be difficult, since the microwave study illustrated that
temperature also effects the sporicidal activity of
radicals.
Effect of Plasma Treatment Pressure on Sporicidal
Activity of Hydrogen Peroxide and RF Discharge
The effect of plasma treatment pressure was determined
at the same HgO^/plasma conditions as used in the previous
section but with 0.5» 0.75» 1»0 and 1.5 torr plasma treat­
ment pressure.
Tests were conducted on Tyvek-packaged B.
subtilis (var. globigii) spores on paper discs.
The
results (Table 12) show that total deactivation can be
achieved at 1.0 or 1.5 torr pressure with 5 minutes of
plasma, and the optinum sporicidal activity was obtained
at 1.0 torr pressure with plasma.
The temperature of the
system increased with increasing pressure, while the
residuals decreased with increasing pressure.
The major difference between this pressure effect and
the pressure effect observed in Figure 23 is due to the
amount of H 202 available on the paper discs.
In this study
the pressure of the system during the pretreatment period
was the same in each test.
The pressure difference only
occurred during the plasma treatment time.
In the pressure
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4
TABLE 1 2
EFFECT OF PRESSURE ON HgOg RESIDUAL, TEMPERATURE AND SPORICIDAL ACTIVITY
WITH TYVEK-PACKAGED BACILLUS SUBTILIS (VAR. GLOBIGII) SPORES ON
PAPER DISCS WITH 15 MINUTES OF 5.0 MG OF H~0„ PRETREATMENT
Sporicidal Activity
Residual
after
(s/so )
Pressure
Temperature ( C)
before and after
15 min. Plasma
15 min.
(torr)
3 min.
5 min.
10 min.
15 min.
plasma
plasma
plasma
plasma
Plasma
(/ig)
initial
final
A t
1.5
5.3xl0~2
0
0
0
19.53
19.4
75.8
56.4
1.0
1 .OxlO-5
0
0
0
70.65
24.7
78.5
53.8
0
91.10
22.7
71.6
48.9
103.38
24.5
56.5
32.0
0.75
-
8.4x10"^
5.3xl0-6
0.5
-
3.0xl0"2
2.3xl0-3
l.OxlO"3
o
106
effect on sporicidal activity in Figure 23 the pressure was
changed during both the HgOg pretreatment and plasma treat­
ment period.
This would effect the amount of H 202 present
on the paper discs.
When the amount of H 202 is the same,
the optimum pressure for plasma treatment shifts from 1.5
torr to 1.0 torr.
The greater sporicidal activity at the lower pre­
ssure’with the HgOg/plasma system may be due to the longer
life times of the radicals formed by the H 2C>2 in the RF
discharge.
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CONCLUSION AND D ISC U SSIO N OF RF DISCHARGE
Of the chemicals evaluated in an RF discharge only
HgOp exhibited significant sporicidal activity.
To better
understand the mechanism involved in the sporicidal activi­
ty with HgOg and plasma the various parameters of the
system were evaluated in detail.
The results of these
studies are discussed below.
Temperature Measurement
The temperature of articles exposed to the plasma
not only depends on the plasma type (continuous or pulsed),
exposure time, power applied, and frequency used to gene­
rate the plasma, but also appears to depends on the surface
to volume ratio of the object and the heat capacity of the
material.
The Fluoroptic thermometer provided a very good
method of measuring the temperature of objects exposed to
an RF plasma, if the object was large enough to provide
easy contact with the thermometer probe.
The temperature
of fibers with very large surface to volume ratios such as
polyethylene microfibers was determined by examining the
heat history of the polymer using Differential Scanning
Calorimeter.
The results at 1.5 torr indicated that the
•«
XUf
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1
r\ P
X w
temperature reached by the Fluoroptic thermometer probe is
very closed to the temperature experienced by very small
fibers.
Dissociation of
with RF Discharge
Hydrogen peroxide is used as the precursor for the
active species generated in the RF discharge.
Time is
required to allow the H o0o to diffuse through the package
b
fas
and contact the spores prior to the application of RF
energy.
In this way, the active species can be generated
on the spore surface.
Tests conducted in which the plasma
was ignited when the HgOg was first introducted into the
chamber indicated that the power required to decompose
HgOg is between
75 and 100 watts of 1:2 pulsed plasma.
If
HgOg was decomposed before it could diffuse to the site of
spores, i.e., by exposure to 100 watts of 1:2 pulsed plasma
during the HgOg pretreatment period, then all sporicidal
activity was lost.
This is consistent with the results
of the microwave discharge studies that showed that radi­
cals cannot diffuse through packaging in sufficient number
to be sporicidal.
Frequency Effect
The frequency used to generate the plasma has been
demonstrated, to have a definite effect on both temperature
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
and sporicidal activity.
For a given coil, the temperature
and sporicidal activity increased with increasing frequency.
For a given frequency, the temperature is higher and spori­
cidal activity is better with the RF coil which has more
turns.
This may be due to more RF power being coupled into
the plasma when a higher frequency or a coil with more
turns is used.
If one assumes that the plasma temperature is a mea­
sure of the induced voltage, then a plot of sporicidal
activity against frequency at a constant temperature indi­
cates (Figure 28) that sporicidal activity increases with
increasing frequency at the same induced voltage.
This
may be due to RF becoming more of a surface effect at
higher frequencies and/or plasma being generated in more
inaccessable areas at higher frequencies.
Pressure Effect
The temperature of the RF plasma increased with in­
creasing pressure at a given frequency.
Apparently, more
RF power is coupled into the plasma as the pressure in­
creases.
The sporicidal activity of the HgOg/plasma system,
however, peaks at 1.0 torr pressure over the pressure range
evaluated (0.5 to 2.0 torr) when the same HgOg pretreatment
conditions were used.
The decreased sporicidal activity
observed at pressures higher than i.O torr could be the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
110
results of reduced electron energy and/or reduced life
times for radicals generated in the plasma.
Since RF
plasma is easier to generate and is more expanded at lower
pressures the optimum sporicidal activity at 1.0 torr pre­
ssure may relate to the ability to generate plasmas in
inaccessabie areas and thus a more uniform radical concen­
tration over the entire sample being radiated.
Sporicidal Activity of H ^O^/Plasma with Aerobic
and Anaerobic Spores
The sporicidal activity of a 0.03 torr vacuum, air
plasma, HgOg vapor and H 202/plasma on both aerobic and
anaerobic spores were evaluated.
The results of this study
showed that no significant kill occurred with vacuum alone.
The aerobic, radiation resistant spore B. pumilus was the
most resistant to air plasma, while the anaerobic spore
C. sporogenes was.the least resistant.
also most sensitive to
C. sporogenes was
vapor alone, which is consis­
tent with the fact that anaerobic organisms have not deve­
loped defense systems to deal with oxidizing chemical
species.
B. subtilis (var, globigii) and B. subtilis were
found to be the most resistant spores to the HgOg/plasma
system.
At the condition required for total kill with
Tyvek-packaged B. subtilis (var. globigii) and B. subtilis
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
I ll
spores on paper discs, no appreciable sporicidal activity
was noted with vacuum alone, plasma alone, and H 202 alone
but total kill (i.e. S/SQ < 1 0 " 6) was obtained with the
combination of H 2C>2 and plasma.
This strong synergism
must relate to the reactive species generated in the
H 202/plasma system.
Mechanism of Action
According to the microwave discharge study, the
hydrogen, oxygen, hydroxyl, and hydroperoxyl radicals are
all sporicidal active species-
However, when H 2C>2 , HgO,
H , 0,, and N ?0 were evaluated for sporicidal activity in
2
2
^
an RF discharge only the H 202/plasma system exhibited any
significant activity.
The heat generated in the RF discharge at the condi­
tion investigated should not kill spores, but it can en­
hance the sporicidal activity of HgOg.
However, this study
indicates that the major activity is not the result of heat
and H 909, but appears to be due to the radicals generated
by the H 2C>2 and plasma.
It is not possible to identify
what specific species are involved in the inactivation of
the spores under the experimental conditions available
during this research.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
112
If the hydroxyl radical generated from H 202/plasma
is the only active species, then the hydroxyl radical gene­
rated from HgO/plasma should also kill spores.
Although
the same bluish plasma was observed with HgO/plasma as the
HgOg/plasma, no enhancement was obtained with HgO/plasma
over the air/plasma even at very high powers.
The bond dissociation energy of N20 to generate
oxygen atom is less than the bond dissociation energy of
HgOg to generate hydroxyl radical.
If 150 watts of pulsed
plasma can dissociate H 202 , then it should be able to gene­
rate oxygen atom from NgO.
Slightly better sporicidal acti­
vity was obtained with NgO/plasma than either 02/plasma or
air plasma, but it does not compare to the total kill ob­
tained with H 202/plasma.
Apparently, the HgOg/plasma generates some sporicidally active species that the other plasmas do not possess.
It is believed that hydroperoxyl radical, the product from
the reaction between hydroxyl radical and hydrogen peroxide,
is the most likely species causing the deactivation of the
spores.
As pointed out by Foner,
within milliseconds the
predominant radical in the H 2C>2 and RF discharge is the
hydroperoxyl radical.
It is a],so possible that a strong
synergism for inactivation of spores exists between the
radicals formed from the dissociation of HgOg and H 202 it­
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
113
self.
In the absence of direct spectroscopic evidence,
it is impossible to provide unequivocal evidence of the
actual active species and/or the inactivation mechanism for
the spores.
Most researchers believed that the hydroxyl radical
is biologically the most damaging species^"’^ .
The results
of this research indicate that in the H 202/plasma system
the hydroperoxyl radical may also play an important role
in sporicidal activity.
Activity from hydroperoxyl radical
would be consistent with the observations of Ewing,
2i
who
suggested that *02 and/or *HC>2 are responsible for the spo­
ricidal activity observed when HgO or HgOg solutions are
'irradiated with x-rays.
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APPENDIX A
MICROBIOLOGICAL WORK
114
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The detailed microbiological work including prepara­
tion of stock cultures, inoculation of substrates, and
assay for survivors of the spores are described below.
Preparation of Stock Cultures
Growth Condition.
Spores were grown for 2k hours
with appropriate broth culture and temperature in an erlenmeyer flask (Table 13)=
Incubation Condition.
Three (3) ml of growth solu­
tion from above was transferred into a petri dish, filled
with appropriate agar, and incubated at appropriate tem­
perature and time (Table 13)=
Wash Condition.
The growth from the agar surface was
scraped off with L-shape glass rod and washed several times
with appropriate solution (Table 13)=
Reconstitution Condition.
The pellet was reconsti­
tuted after washing in an appropriate solution (Table 13).
Inoculation of Substrates
7
The stock solution was diluted to about 5x10' orga­
nisms/ml in an appropriate solution, 0.05 ml was added onto
each substrate, and overnight drying time was allowed
before use (Table 13)=
115
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE 13
PARAMETERS FOR MICROBIOLOGICAL WORK
test organism
growth
condition
for stock
culture
incubation
temp.
B.
C. sporogenes
(°C)
broth media
37
30
30
polypeptonepeptone
nutrient broth
nutrient broth
anaerobic agar nutrient
agar media
B . subtilis
T v a r . globi gii
subtilis
#
agar
nutrient
B.
pumilus
30
*
agar
tryticase
soy broth
tryticase
soy agar
, condition
for stock
culture
wash
condition
for stock
culture
time (days)
temp.
(°C)
number
of wash
solution
5
h
3
7
7
8
37
30
30
30
3
2
2 o saline h 2 o
reconstitution condi­
H-0% ethanol
tion for stock culture (not denatured)
inoculation condition
bOfo ethanol
for substrates
(not denatured)
diluent
water
solution
plate
agar media
anaerobic agar
5
2
3
3
5
time (days)
temp.
(°C)
3
saline h 2°
water
40 fo ethanol
(not denatured)
^ 0 % ethanol
(not denatured)
40 % ethanol
(not denatured]
k o % ethanol
(not denatured]
water
water
water
water
"w" media
"w" media
h
2°
saline
h
2o
h
2°
count
condition
2
water
tryticase
soy agar
3
3
3
2
37
30
30
30
* : Included added MgSO^ to enhance sporulation
: See references Si and 82
Assay for Survivors
The sporicidal activity was tested hy the plate count
test method.
This test method gives quantitative data and
is widely used for the rapid screening and evaluation of
various
test conditionss
The plate count test used a
serial dilution technique and gives an approximate count
of the number of colony forming units present after expo­
sure to the test conditions.
The procedure are described
below.
(a) Stripping fluid was prepared by adding 0.4- g of pota­
ssium phosphate (monobasic), 10.1 g sodium phosphate
(dibasic), and 1.0 g triton x-100 to 1 liter of water.
(b) Item A:
Eight (8) mg of 2000-5000 sigma unit catalase
81
(sigma corp. c-10 catalase) was added to 300 ml stri­
pping fluid.
(c) Item B:
(i) For paper disc : The paper disc was vortexed in 10 ml of solution from item A
with 5 glass beads in a test tube.
(ii) For glass slide s The glass slide, in 10
ml of solution from item A, was rubbed
with a rubber policeman in a petri dish.
(d) Item C:
One (1) ml of solution from item B was placed
into 9 ml of appropriate diluent solution (Table 13)«
(e) Item D:
One (1) ml of solution from item C was placed
into 9 ml of appropriate diluent solution (Table 13)»
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
lib
(f) Item Es
One (1) ml of solution from item D was placed
into 9 ml of appropriate diluent solution (Table 13).
(g) The appropriate agar media was added to 1 ml solutions
from items B, C, D, and E and they were incubated at
the appropriate temperature and time (Table 13).
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APPENDIX B
REPRODUCIBILITY OF THE
MICROBIOLOGICAL TEST RESULTS
119
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
120
The plate count test was the method used in this re­
search to evaluate the sporicidal activity at various test
conditions.
It is "based on a serial dilution technique.
Since this method gives an approximate count of the number
of colony forming units present after the tests, a discu­
ssion concerning the reproducibility of this method is
presented below.
Microbiological test results on the effects of pre­
ssure (Figure 23) and frequency (Figure 26) on the sporici­
dal activity of HgOg/plasma are presented in Tables 14, 15
and 16.
The largest standard deviation of S/SQ in Table
14 is 1.1 (only the mantissa of the number is considered)
with the average value about 0.4.
The largest standard
deviation of S/SQ in Table 15 is 1.7 with the average value
about 0,5,
The largest standard deviation of S/SQ in Table
16 is 3*4 with the average value about 1.0.
The average
standard deviation of all three Tables is about 0.7.
It
is quite normal to have this kind of standard deviation in
microbiological results.
After considering the standard
deviation for each test, the effects of pressure and fre­
quency on sporicidal activity can still be easily deter­
mined .
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
MICROBIOLOGICAL TEST RESULTS OF DATA PRESENTED IN
FIGURE 23 - EFFECT OF PRESSURE
Number of Surviving Spores (S)
Pressure
Control
s/s0
(torr)
<So>
Sample 1
0.5
3.4x10^
9.9x10
1.0
2. 7x10'*
1.5
2.0
Sample 2
Average
1.8xl05
1 .4xl03±0.4xl03
4,,lxl0“1±l.lxl0"1
3.1xl03
3 .4xl03
3 . 3 x 1 0 3± 0 . 2 x 1 0 3
1 „ 2 x 1 0 -2± 0 . 1 x 1 0 -2
3.4 x 105
0
0
0
0
6.0xl05
1.2xl02
l.lxlO2
1 . 2 x 1 0 2+ 0 . 1 x 1 0 2
2•0xl0~^i0.2xl0-^
4
1 ?1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE 1 4
MICROBIOLOGICAL TEST RESULTS OF DATA PRESENTED IN FIGURE 26
- EFFECT OF FREQUENCY WITH 12 TURN COIL
Frequency
Control
Number of Surviving Spores (S)
s /s 0
(MHz)
'V
1.09
Sample 1
Sample 2
Average
3.7xl06
1.2xl05
4.9x10^
8.5x10^+3.6x10^
2.3xl0"2+1.0xl0“2
1.29
3.7xl06
1.4xl03
1.6xl02
7 •8 x 1 0 2± 6 .2xl02
2.1xlO“Zf±1.7xlO“Z|’
1.49
3*7xl06
5.OxlO1
1 .OxlO2
7 . 5 x 1 0 1± 2 . 5 x 1 0 1
2 . 1 x 1 0 " 5± 0 . 7 x 1 0 ~ ^
1.69
3.7xl06
5.OxlO1
5.oxio1
5.0xi01±0
1 . 4 x 10~-5± 0
1.89
3.7xl06
1.2xl02
1.2xl02
1 . 2 x 1 0 2± 0
3 . 2 x 1 0 " 5±0
2.09
3.7xl06
8 .OxlO1
4.OxlO1
6.OxlO1! 2.OxlO1
1 . 6 x 1 0 " 5+ 0 . 5 x 1 0 " 5
2.29
3.7xl06
6.OxlO1
5.OxlO1
S.Sxlo^o^xlo1
l ^ x l O ’^+O.lxlO"-5
2.49
3.7xl06
5.OxlO1
5.oxio1
5.0xlo1±o
1.4xl0“^±0
122
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE 15
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE 16
MICROBIOLOGICAL TEST RESULTS OF DATA PRESENTED IN FIGURE 26
- EFFECT OF FREQUENCY WITH 6 TURN COIL
Frequency
Control
Number of Surviving Spores (S)
s/so
(MHz)
< V
Sample 1
Sample 2
Average
2.09
3.8x 106
1.5xl06
8.1x10"*
1.2xl06+0.4-xl06
3«2xlO-1+l.lxlO”1
2.29
3.8x 106
5.7x10^
3.8x10^
4-. 8x10^+1.0x10^
1„3x 10-2+0.3x 10“ 2
2.4-9
3.5x 106
4-.0x10^
1.6x10^
2.8x10^11.2x10^
8 „ 0 x 1 0 ~ 3 +3.4-x 1 0 “ 3
2.69
3.5x10^
3.7xl03
4-.6xl03
4-.2x10 3+ 0. 5x10 3
1„2x 1 0 “ 3! 0 . 1 x 1 0 ~ 3
2.89
3.5xl06
l.lxlO2
9.OxlO1
1.0xl02± 0 .lxlO1
2 . 9 x 10"-5+ 0 , 1 x 1 0 “ 3
3,09
3.5xl06
2.OxlO1
6.OxlO1
4-.OxlO1! 2. OxlO1
1.lxl0_3+ 0 .6xlO~3
3.29
3.1xl06
1 .OxlO1
2.OxlO1
l.5xlo1+o.5xio1
4.3xlO“6+1.4-xlO-6
3.4-9
3.1xl06
0
0
0
0
3.69
3.1xl06
l.OxlO1
0
o •5xlo1+o•5X101
1.4-x 1 0 _ 6 +1.4-x 1 0 " 6
3.89
3.1xl06
1 .OxlO1
0
o^xio^o^xio1
1 ,4-xlO“6+1.4-xlO-6
REFERENCES
1. Russell,A.D., Chemist Aerosol News, 1965, 36, 38.
2. Sussmann,A.S.; Halvorson.H.O., "Spores, Their Dormancy
and Germination", 1966, Harper & Row, New York.
3. Block,S.S., "Disinfection, Sterilization and Preserva­
tion", 1983» lea & Febiger, Philadelphia.
k.
Bruch,C.W.; Bruck,M.K., "Sterilization", 1971, Mark
Publishing Co.
5. Sykes,G., "Disinfection and Sterilization", 1958,
Chapman & Hall, London.
6 . Kiefer,J., J. Theor. Biol., 1971* 30, 307.
7 . Ho.S.K.; Ho,Y.l., Radiat. Res., 1972, 51, 1^2.
8 . Varghese,A.J.; Day,R.S., Photochem. Photobiol., 1970,
11, 511.
9. Pollard,E.C.; Tilberg,A., J. Biophys., 1972, 12, 133.
10. Bovey,F.A., "The Effect of Ionizing Radiation on Nature
and Synthetic High Polymers", 1958, Interscience
Publishers, New York.
11. Woodroof,E.A., J. Bioeng., 1978, 2, 1.
12. Bruch,C.W.}Bruch,M.K., "Gaseous Disinfection” , 1970,
Marcel Dekker, New York.
13. Gill,J.R.; Bruch,C.W., MD&DI, 1983, October, 36.
LC*
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
14. Altman,K.I.; Gerber,G.B.; 0kada,S., "Radiation Bioche­
mistry", Vol. I, Cells, 1970, Academic Press, New York.
15. Ejdus,A.K., "Physichemical Principles of Radiobiologi­
cal Process and of Protection against Radiation",
1972, Atomizdat, Moscow.
16. Grecz,N.; Schgal,L.R., FAO/lAEA, 1970, 5 , 11.
1?. Friedman,Y.S.; Grecz,N., Acta Alimentaria, 1974, 3 , 251.
18. Power,E.L.; Cross,M., International J. of Radiation
Biology, 1970, 17, 501.
19. Edwards,H.E.; Navaratnam,S.; Parsons,B.J.; Phillips,
G.O., Stud. Phys. Thero. Chem., 1979, 6 , 283.
20. Feldberg,R.S.; Carew,J.A., Int. J. Radiat. Biol., 1981,
40(1), 1 1 .
21. Ewing,D., Radiation Research, 1982, 92, 604.
22. Ewing,D., Radiation Research, 1983, 96, 275.
23. Ewing,D., Int. J. Radiat. Biol., I983, 43(5), 565.
24. Ewing,D., Radiation Research, 1983, 94, 171.
25. McCord,J.M., Surgery, 1983, 9» 412.
2o. McCord,J.M.; Day,E.D.Jr., FEBSLett., 1978, 86, 139.
27. Winterbourn,C.C., J. Biochem., 1979» 182, 625«
28. CoHen,G., "Superoxide and Superoxide Dismutases",
1977» Academic Press, Loridon.
29. Fridovich,I., Annu. Rev. Biochem., 1975* 44, 147.
30. Lesko,S.A.; Lorentzen,R.J.; Ts'0,P.0.P., Biochemistry,
1980, 19, 3023.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
31. Ananthaswamy,H .N .j Eisenstark,A., Photochemistry and
Photobiology, 1976, 24, 4 3 9 .
32. Bayliss,C.E.; Waites,W.M., Microbiology Letters, 1979,
5, 331.
33. Bayliss,C,E.; Waites,W.M., J. of Appl. Bacteriology,
1979, 47, 263.
34. Bayliss.C.E.j Waites,W.M., J. of Appl. Bacteriology,
1980, 48, 417.
35* Johansen,I., "Cellular Radiation Biology", 1964,
Williams & Wilkins, Baltimore, Maryland.
3 6 . Johansen,I.5 Howard-Flanders,P., Radiation Re s ., 1965,
24, 184.
37. Block,J.; Luthjens,L.H.; Ross,A.L.M., Radiation Res.,
1967, 30, 468.
38. Repine,J.E.; Pfenninger,O.W.j Talmage,D,W.; Berger,
E.M.j Pettijohn,D.E., Proc. Natl. Acad. Sci., 1981,
78(3), 1001.
39* Ewing,D.5 Powers,E.L., Science, 1976, 194, 1049,
40. Singh,H»; Vadasz,J.A., Int. J. Radiat. Biol., 1983,
43, 587.
41. Michaels,H,B.; Peterson,E.C.; Epp,E.R., Radiat. Res.,
1983, 95, 620.
42. Willson,R.L., Ciba. Found Symp., 1979, 65, 19•
43* Venngopalan,M.; Jones,R.A., "Chemistry of Dissociated
Water Vapor and Related Systems", I968, Interscience
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
12?
Publishers, New York.
44. Jones,R.A,; Chan,W.; Venngopalan,M., The J. of Phys.
Chem., 1969, 73, 3693.
4-5. Venngopalan.M.j Shih,A.f Plasma Chem. and Plasma Proc.,
1981, 1, 191.
4-6. Banlch,D.L.; Drysdale,D.D.? Horne,D.G.; Llofd,A.C.,
"Evaluate Kinetic Data for High Temperature Reaction,
Vol. 1 - Homogeneous Gas Reaction of the
System",
1972, CRC Press, Cleveland, Ohio.
47. Badin,E.J., J, of Am. Chem. Soc., 194-8, 7 0 , 3651 •
4-8, Rodebush,W.H.; Wahl,M.H., J. of Chem. Phys., 1933, 1,
6 96.
4-9. Oldenberg,0., J. of Chem. Phys., 1935, 3, 266 .
50. Jackson,W.F., J. Am. Chem. Soc., 1935, 57, 82.
51. Geib,K.H., J. of Chem. Phys., 1936, 4-, 391 .
52. Frost,A.A; Oldenberg,0., J. of'Chem. Phys., 1936, 4-,
64-2.
53. Rodebush,W.H.; Wende,C.W.J.; Campbell,R.W., J. Am.
Chem. Soc., 1937 , 59, 1924-.
54-. Rodebush,W.H. j Keizer,C.R.; McKee,F.S.; Quagliano, J.V.,
J. Am. Chem. Soc., 194-7, 69, 538*
55. Ureg,H.C.; Dawseg,L.H.; Rice,F.O., J. Am. Chem. Soc.,
1929, 51, 1371 56. Frost,A.A.; Oldenberg,0., J. of Chem. Phys., 1936, 4-,
781
.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
126
5 7 o Foner,S.N., Science, 1964, 143, 441.
58. Bithell,R.M., U.S. Patent 4348357, 1982..
59. Fraser,S.J.; Gillette,R.B.; Olson,R.L., U.S. Patent
3851436, 1974.
60. Fraser,S.J.; Gillette,R.B.; Olson,R.L., U.S. Patent
3948601, 1976.
61. Boucher,R.M., U.S. Patent 4207286, 1980 .
62. Bithell,R.M., U.S. Patent 4321232, 1982.
63. Tsuchida,H«s Honda,K.; Muto,M.; Amano,S.; Naito,S.;
Yazaki,J., Japaness Application Disclosure 103460,
1983.
64. Sworski,T.J.; Hochanadel,C.J.; Ogren,P.J., J. Phys.
Chem., 1980,
84, 129-
6 5 . Volman,D.H., The J. of Chem.
Phys., 1949»
17* 947.
66. Greiner,N.R., The J. of Chem. Phys., 1966, 45, 9 9 .
6 7 . Stief,L.T.; DeCarlo,V.Toi The J. of Chem. Phys., 1969,
50, 1234.
68. Meagher,J.F.; Heicklen.J., J. of Photochem., 1974/75,
3, 455.
69 . Greiner,N.R., The J. of Chem. Phys., 1967, 46, 2795.
70. Wine,P.H.; Semmes,D.H.; Ravishankara,A.R., J. Chem.
Phys., 1981,
75, 4390.
71.
Demore,W.B., The J. of Phys.
72.
Hochanedel,C.J.;Sworski,T.J.; Dgren,P.J., J. of Phys.
Chem., 1980,
Chem., 1979,
83,1113.
84, 3274.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
73« Greiner,N.R., The J. of Phys. Chem., 1968, 72, 406.
74. Barrett,J.J., J. of Opt. Soc. Amer,, 1975, 65, 9 2 .
75* Avizonis,P.V.; Dean,D.R.; Grotbeck, R., Appl. Phys.
Lett., 1973, 23, 3757 6 . Porter,R.A.; Harshbarger,W.R., J. Electrochem. Soc.,
1 9 7 3 t 126, 460.
77. Egerton,E.J.j Nef,A.j Millkin,W.; Cook,W.; Baril,D.,
Solid State Technology, 1982, 84.
78. Luxtron Fluoroptic Thermometer Manual, Mountain View,
CA.
79. gray,A.P., Thermochimica Acta, 1970, vol. 1, No. 6, 563.
80. Horwitz,W., "Official Methods of Analysis of Official
Analytical Chemists", 14th edition, 1984, Washington,
D.C., page 72.
81. Wallen,S.E.; Walker,H.W., J. of Food Sci., 1979, 44,
560
.
82. Wang,D.I.; Scharer,J.; Humphrey,A.E., Appl. Microbiol.,
1964, 12, 451 .
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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