Note: Descriptions are shown in the official language in which they were submitted.
11043Z2
SL~EDED GAS PLASMA STERILI ZATION METHOD
This invention relates to gaseous sterilization
by the treatment of objects or materials with a chemical in
the gaseous or vapor state to destroy all microorganisms
with which they have been contaminated. The need for such
a method of sterilization has developed from the use of many
items that cannot be subjected to heat, radiation, or liquid
chemical sterilization.
In practice, only two gases or vapors have been
commercially used on a large scale for surface sterilizing
purposes and these are formaldehyde vapors and ethylene oxides
gas. However, each suffer from drawbacks.
Formaldehyde vapors have been used as a fumigant
for many decades in the h~spital, agricultural and industrial
fields. The limitations of this technique are numerous.
To kill tough aerobic and anaerobic bacterial spores at room
temperature, one needs at least a 24 hour contact time with
a vapor having at least 70% relative humidity. This type of
vapor is extremely corrosive and the fumes are very irritating.
It is also very difficult to maintain a hîgh level of
formaldehyde gas since CH2O is stable in high concentrations
only at temperatures above 80C in humid air. At ordinary
room temperatures formaldehyde gas quickly polymerizes and
it dissolves readily in the presence of water. Thus gaseous
sterilization with formaldehyde can be regarded as a
I misnomer because introduction of formaldehyde gas into a
closed space serves mainly as a mechanism for distributing
either moisture films in which formaldehyde is dissolved or
2~104;~zz
solid formaldehyde polymers over all available surfaces
within the enclosed space. Very
inConsistent and sometimes contradictory results have been
reported in hospital disinfection, patient rooms, bedding,
etc.,and in agricultural applications such as eggs and
hatcheries sanitizing. Formaldehyde vapor has a very weak
penetrating ability and, if used in an a.tmosphere with
traces of hydrochloric acid, it can quickly produce at 70C
an~ 40% relative humidity) bis-(chloromethyl)-ether, which
is a carcinogenic agent.
To minimize the abovementioned drawbacks in
hospital applications, a new approach was recently developed
which combines the use of subatmospheric steam and
formaldehyde gas at 80C in autoclaves. This method i~s
said to kill most sporulated microorganisms at the concentra-
tion normally encountered in hospital practice while
decreasing the aldehyde residue on instruments. It
requires a time exposure of t-~o hours with a formalin
concentration of 8 gr. per cubic foot of autoclave.
~lowever, despite the long contact time and the relatively
high ~emperature, the method does not satisfy the
stringent requirements of the sporicidal AOAC (Association
of Official Analytical Chemists) test in the United States
of America.
From the foregoing, it is apparent that formal-
dehyde vapors, besides their toxicity and irritating charac-
teristics, are difficul.t to handle at room temperatures and
they do not provide a fast and reliable method to satis-
factorily handle most of the hospital and industrial applica-
30 tions.
,
3 110a~3Z2
In the past two aecades ethylene oxide (ETO) has
become the most popular method to gas sterilize both in
hospitals and industry. While initially ETO seemed an ideal
technique to replace formaldehyde fumigants, very serious
limitations from the toxicity viewpoint have recently
attracted the attention of health authorities.
The average time needed to sterilize medical
instruments in an ETO unit is 180 minutes at 30C., but it
has to be followed by a long de-aeration period. For
instance, the de-aeration time for medical devices is between
2 and 8 hours in a de-aerator machine, but it oscillates
between 1 and 8 days at room temperature. On rubber gloves,
the residues can burn the hands; on tubes carrying blood,
they will damage red blood cells and cause hemolysis. Endo-
tracheal tubes which are not properly aerated can causetracheitis or tissue necrosis.
Besides the risks due to the toxicity of ETO
residues, other accidents have been reported due to the ex-
plosive characteristics of pure ETO. As little as 3%
ethylene oxide vapor in air will support combustion and will
have explosive violence if confined. To solve this problem,
various diluent gases such as CO~ or fluorinated hydro-
carbons have been mixed with ETO in some commercial
formulations.
It is apparent, therefore, that ETO sterilization
became widely used not because it was an ideal sterilant,
but rather since there seemed to be no alternative gas steri-
lant method which was capable of as fast a sporicidal action
without any drawbacks from the toxicological or environmental
: 30 viewpoint.
~0~3Z2
The present invention provides an alternative to
ETO sterilization with the advantages of faster sporicidal
action, no de-aeration period, no toxic residue, and no
explosion risk. Moreover, this invention provides a more
5 economical approach from the running and investment cost view-
point when comparing the volume of material treated per unit
of time.
In accordance with the present invention, there is
provided a method of sterilizing a surface comprising con-
10 tacting the surface with a low temperature gas plasma contain-
ing at least 10 mg/l of an aldehyde under a subatmospheric
pressure.
The term "Sterilization" as used herein refers to
sporicidal action against Bacillus subtilis ATCC (American
15 Type Culture Collection) 19659 and Clostridrium sporogenes
(ATCC 3584) because they are the resistan~ microorganisms used
in the fumigant-sterilant test according to the requirements
of the AOAC (Official Method of Analysis of the Association
of Official Analytical Chemists, 12th ed., Nov. 1975).
20 The destruction of these two resistant species of spores by
the AOAC procedure leads automatically to the destruction of
other less resistant microorganisms, such as, mycobacteria,
non-lipid and small viruses, lipid and medium size viruses,
and vegetative bacteria.
A better understanding of the cidal mechanism of
a low temperature gas plasma in accordance with this invention
may be had by consideration of the physical structure of a
highly resistant spore. Figure 1 represents the typical
structure of a typical bacterial spore. The typical bacteria
30 spore is surrounded by an exosporium which is a loose sac
110~3Z2
peculiar to so~e spores species, and possesses, from the
outside to the inside, successively, (a) multi-layered coats
containing disulphide (-S-S-) rich proteins, (b) a thick
cortex layer which contains the polymer murein (or peptido-
5 glycan), (c) a plasma membrane, and (d) a core or sporeprotoplast.
The first line of resistance of the spore to exo-
genous agents consists of the proteinaceous outer coats which
contain keratin-like proteins. The stability of keratin
10 structures is due to frequent primary valence cross links
(disulphide bonds) and secondary valence cross links (hydrogen
bonds) between neighboring polypeptide chains. Keratin like
proteins are typically strong, insoluble in aqueous salt
solutions or in diluted acid and basic solutions, and are
15 resistant to proteolytic enzymes and hydrolysis. The layered
outer coats, thus, are rather inert and play a predominant
role in protecting the spore against exogenous agents. They
seem to play an important role in cidal action through
physical or chemical modifications which affect the diffusion
20 of cidal molecules, excited atoms or radicals inside the
microorganism protoplast.
To alter the multilayered outer coats and thus allow
further penetration and possible interactions in the critical
cortex or protoplast regions, a very active agent mus be
25 chosen, and it has been found that an ionized gas plasma is
an excellent vehicle to provide reactiYe atoms, free radicals,
and molecules which will drastically alter the protective
layers of bacteria, fungi, and spores. The presence of
small amounts of aldehyde vapors in the ionized low tempera-
llOi3~2
ture non-oxidizing gas plasma, in accordance with this inven- I
tion, leads to the destruction of sporulated and non- ¦
sporulated microorganisms.
In the present invention, the objects to be decon-
5 taminated are exposed to a continuous flow of low temperaturegas plasma seeded with a small amount of an aldehyde, usually
an aromatic, heterocyclic, saturated or unsaturated aldehyde.
The gas plasma is a partially ionized gas composed of ions,
electrons, and neutral species.
The low temperature gas plasma is formed by gaseous
electric discharges. In an electrical discharge, free elec-
trons gain energy from the imposed electric field and lose
this energy through collisions with neutral gas molecules.
The energy transfer process leads to the ormation of a
15 variety of highly reactive products including metastable
atoms, free radicals, and ions.
For an ionized gas produced in an electrical dis-
charge to be properly termed a "plasma", it must satisfy the
requirement that the concentrations of positive and negative
20 charge carriers are approximately equal. The plasma used in
the present invention are glow discharges plasma and are also
termed "low temperature" gas plasma. This type of plasma is
characterized by average electron energies of 1 to 10 eV and
electron densities of 109 to 10 2 per cm . Contrary to the
25 conditions found in arcs or plasma jets, the electron and
gas temperatures are very different due to the lack of thermal
equilibrium. In a glow discharge, the electron temperature
can be ten to a hundred times higher than the gas temperature.
The latter property is important when sterilizing the surfaces
30 of thermally sensitive materials.
, .
7 :1104322
r In the low temperature gas plasma used in this
invention, there can be distinguished two types of reactive
elements, i.e. those which consist of atoms, ions or free
radicals and those which are small high energy particles
5 such as electrons and photons. In glow discharges a large
amount of ultraviolet radiation (UV) is always present.
The UV high energy photons (3.3 to 6.2 eV) will produce
strong cidal effects because they correspond to a maximum of
absorption by DNA (deoxyribonucleic acid) and other nucleic
10 acids~ However, in the case of spores which can reach one
millimeter in diameter, photon energy can be quickly dissipa-
ted through the various spore layers and this may restrict
photochemical reactions to outer coats. The photon energy is
rather restricted to thin layer surface modifications and is,
15 therefore, more efficacious when dealing with the smaller non-
sporulated bacteria. In the case of high resistance spores,
the photonic action may contribute to partial alteration of
the disulphide rich proteins coat and thus facilitate the
diffusion of free radicals, atoms, or excite~molecules inside
20 the core region.
In the present invention, small amounts of vaporized
aldehyde monomers and free radicals present in a low tempera-
ture gas plasma can greatly increase the overall biocidal
action of a gas plasma.
The exact mechanism whereby enhanced sporicidal
activity is attained using the aldehyde-seeded plasma stream
is not fully understood, but some mechanis~ can be considered.
For example, due to the presence of atomic or excited oxygen
in the gas phase, the aldehydes may produce short life very
30 reactive epoxides and other intermediates and free radicals
~10~322
which may interact with many proteins and nucleic acids groups
in outer layer coats and thereby improve diffusion of cidal
groups.
The next possible step in the diffusion of cidal
5 groups is the penetration inside the cortex layer whose
major component is the polymer murein (or peptidoglycan).
Murein is a large, crosslinked, net-like molecule. A con-
jugat~d attack by atomic oxygen and aldehyde radicals on
the polymer rapidly shakes and modifies the tight polymer
10 structuxe of the cortex layer, leading to its destruction.
In addition, there is the potential for alteration
of the hypothetical pathway of dipicolinic acid synthesis by
the aldehydes. It has long been speculated that since calcium
and dipicolinic acid (DPA) occur in spores in roughly equimolar
lS amounts, they form a salt complex whose role is capital in
spores resistance. The exact location of the calcium salt
in spores is a problem which remains to be solved. The fast
access of aldehydes into the cortex, mainly a result of gas
plasma oxidation, may help blocking the amine groups of the
20 aspartic ~-semialdehyde thus interfering directly with the
DPA synthesis.
The latter mechanism may explain why short exposures
to a plasma gas in the presence of aldehydes can quickly
destroy spores or their germinating capabilities. The
25 aldehydes seeding method of the present invention results in
a shorter contact time in gas plasma to achieve a sporicidal
effect, as compared with other gaseous phase sterilization
procedures.
The cidal action of the low temperature aldehyde
9 ~ 32~ ~
seeded gas plasma is so fast sometimes, for example, less than
ten minutes, that the possibility of inducing reactions
inside the core or protoplast is rather small. The central
portion of the spore is functionally a vegetative bud, which
5 contains the heriditary charter, a repressed protein synthe-
sizing system, the enzymes necessary to initiate the synthesis
of new enzymes and structural materials, and, presumably,
reserves for the supply of energy intermediates. The modifi-
cations taking place in the outer coats, cortex and plasma
10 membranes are sufficient to fully explain the cidal results
obtained with the present invention. References above to the
oxidation phenomena in a gas plasma are not restricted to the
use of pure oxygen as an ionized gas but also includes the use
of oxygen-containing gases like air, carbon dioxide and N2O.
15 Although not as fast as oxidizing plasmas, a noble gas, such
as argon or helium, or nitrogen plasmas, can be seeded with
aldehydes to decrease sterilization time.
The present invention, therefore, enables a con- ¦
siderable re~uction in sporekilling time to be achieved over the
20 values observed in conventional oxidizing and non-oxidizing
gas plasmas. While excited ions, gas molecules, and photons
modify the protective layers of the spores, active aldehyde
radicals penetrate the changing structures and initiate many `
additional lethal reactions which accelerate the killing
25 process. A faster surface sterilization time results in a
more economical process and provides the possibility to handle
many highly heat sensitive materials, which may be degraded by
prolonged exposure to the gas plasma, even at high tempera-
tures below 100C. No severe corrosive or toxic residuals
30 are observed when adding aldehydes to a gas plasma.
1~0~3ZZ
To produce a gas plasma of the type required in the
present invention, the carrier gas may be excited by one of
two different radio-frequency methods. The first approach
consists of a ring type or inductive discharge technique,
5 while the second method consists of a parallel plate or capa-
citive discharge technique. The processing area consists
always of a glass, plastic, or aluminum chamber maintained
under subatmospheric pressure, generally 0.1 to 10 mm. of
mercury, into which a controlled flow of gas and aldehyde
10 vapor is constantly moving under the continuous suction of a
vacuum pump. To excite gases and vapors in the processing
area, the radio-frequency energy delivered by a generator
is coupled through an inductive coil wrapped around the pro-
cessing chamber or by means of capacitive discharge plates
15 placed outside the chamber or chamber entrances. While in
operation, the RF ~radio frequency) discharge glow can be
made to extend virtually throughout the entire processing
chamber. In some instances, the electrodes may be positioned
in the processing chamber.
There are many ways to design electronic circuitry
for maximizing RF energy coupling into the discharging gas.
Energy coupling optimization, which can reach up to 90%, can
be achieved by matching the gas load impedance to the im-
pedance of the ampli~ier plate output circuit and the tank
25 coil. The best impedance matching is achieved by a tuning
process which consists of adjusting variable condensers in a
low impedance matching network connected by coaxial cables
between the reactor chamber and the generator. In more modern
designs, the processing chamber and the relatively low power
110'132~ 1
11 i
generator are coupled directly through high impedance connec-
tors. This eliminates the complicated low impedance network
and simplifies the electronic package. During power coupling
to the gas plasma, a small amount of power is always lost
5 due to heating effects. There is also an amount of power
reflected back to the generator. To know how efficiently one
is discharging energy in the gas, a RF wattmeter is often in- !
serted in the electronic circuit to monitor the difference
between forward and reflected power.
10Gas plasma generators operate generally around 13.5
Megahertz (MHz~ but frequencies in the range of 1 to 30 MHz
also are satisfactory, and even may range up to 100 MHz.
The gas plasma may also be formed a~ higher fre-
quencies in the microwave region, with frequencies ranging
15 from 100 to 300,000 MHz. A preferred microwave frequency
from the practical viewpoint is 2450 MHz. In the microwave
region, the atomic or excited molecular species have a longex
life time than those formed at radio frequencies and they can
persist downstream quite a distance into the glowless region.
20 This is an advantage from the analytical viewpoint, but it
is also balanced by the more complicated and, therefore, more
expensive electronic circuitry required. When using microwave
gas excitation, the processing chamber is usually designed as
a cavity, the generator is generally a magnetron type device
25 and the electro-magnetic energy is conveyed by standard wave
guides.
Irrespective of the gas excitation frequency, it has
been observed that the presence of small amounts of aldehyde
vapors in the gas plasma considerably reduces the time
110~322
12
needed to kill sporulate~ and non-sporulated bacteri-a;
The invention is described further, by way of illus-
tration, with reference to Figures 2 to 4 of the accompanying
drawings, wherein:
Figure 2 is a schemati~ representation of an appara-
tus for sterilizing various hospital type disposals in a
semi-continuous manner;
Figures 3 and 3A are sectional views of the
sterilizing chamber of Figure 2; and
Figure 4 is a schematic representation of an alter-
native form of sterilizing chamber using microwave frequencies.
Referring to the drawings, Figure 2 illustrates the
elements of a low temperature seeded plasma (referred to later
as LTSP) system used for sterilizing in a semi-continuous
15 manner various hospital type disposals. The system comprises
a tunnel-like processing chamber 1, having a door 2 at each
end, only the door 2 at the left-hand entrance side being
shown. The disposables or non-disposables, for instance,
pla.stic bottles of parenteral orophthalm~lo~ical solutions,
20 are loaded in the cylindrical tunnel chamber by means of a
conventional automatic rail conveyor type system ~not shown).
After loading, the front and rear doors 2 are closed auto-
matically by means of an electrically driven mechanical system
3. The loaded tunnel processing chamber 1 is then subjected
25 to vacuum to provide a subatmospheric pressure therein by
means of a vacuum line system 4 connected to a trap 5 and to
a vacuum pump 6. The subatmospheric pressure is generally
about 0.1 to 10 mm. of mercury inside the entire processing
chamber 1.
1104322
13
The gas to be ionized then is delivered from a com-
pressed gas line or bottle 7, the pressure and flow rate being
regulated by pressure gauges and by a constant flow rate
membrane or needle valve 8. Aldehyde vapors are added to the ,
5 gas flow from a container 9 by allowing the gas to bubble
through liquid aldehyde and entrain the aldehyde vapors. A
flowmeter 10 is inserted between the aldehydes container 9
and the inlet into the tunnel chamber 1. The mixture of gas
and vapor is delivered through a hollow pipe line 11 with
10 numerous small holes properly spaced for an even distribution
into the tunnel chamber.
After evacuating most of the air in the tunnel
chamber 1, the gas/vapor mixture is released in the processing
area. The gas/aldehyde vapor flow is adjusted according to
15 the size and volume of the tunnel 1. The plasma formation is
then initiated by proper impedance matching with inductive
and capacitive controls, using an RF coil 12 which is part of
an electrical circuit comprising a matching network 13, a
power wattmeter 14 and an RF generator 15 converting AC
20 (alternating current) standard current into 13.5~ MHz high
frequency. The RF generator 15 used for sustaining a plasma
discharge should be capable of withstanding large variations
in the load impedance, and essentially comprises a DC (direct
current) power supply~ a crystal controlled RF oscillator and
25 a solid state buffer amplifier. Final amplification is
accomplished by a power amplifier designed around a power
tube to accommodate large variations in load impedance.
According to the typeof installation, a single inductive
coil extending over the entire tunnel length may be driven
s, .,
32Z
14
from a single power generator or a series of smaller coil
sections may be operated from smaller modular type RF genera-
tors.
During RF excitation, continuous removal of gas
5 plasma flow is effected over the reaction time period
required to achieve complete sterilization, usually between
5 and 20 minutes. The RF excitation is then automatically
shut down, the gas flow is interrupted and the vacuum pump is
stopped. Air is introduced automatically in the tunnel
lO chamber 1 by a two-way valve 16. The two end doors are
electro-mechanically opened and the samples container is
automatically pulled out from the tunnel on a railing sliding
system. The tunnel chamber l is then ready for sterilizing
a new load. The entire sterilization cycle time generally
15 takes between lO and 30 minutes according to the type of
processed material and power output level.
Figures 3 and 3a are more detailed sectional views
of a longitudinal and lateral cross section, respectively, of
a sterilizing tunnel type processing chamber 1, 2S shown in
20 Figure 2. The tunnel 17 is of cylindrical shape around a
main axis and essentially consists of two concentric cylin-
drical pipes 18 and l9 made of highly resistant inert material,
such as, glass or a polymeric material, for exar;lple, a
polysulfone, which are held by compression on end flanges
2~ with silicone type O-rings 20. After assembling the internal
pipe l9 inside the external pipe 18, a hollow space ring 21 is
created in which vacuum and subatmospheric pressure is pro-
vided by vacuum pump suction through bottom openings 22. To
permit a subatmospheric atmosphere to be formed around the
110~32Z
objects to be decorltaminated, slots or holes 23 are perforated
at the bottom of the internal cylinder 19. The objects to be
sterilized, for example, plastic bottles 24 of parenteral
solutions, are placed in a basket of parallelepipedic shape
5 25, which slides over a rail track 25 on roller bearing
equipped wheels 27. At the beginning of the sterilizing
cycle, the front and end doors 28 and 29 are automatically
opened by an electrically operated device 30 which rotates the
door lB0 around the hinge 31. The front and end doors of
10 the tunnel are generally made of a dark ultraviolet absorb-
ing polymeric material to prevent the dangerous photon
emission from escaping from the chamber while allowing its
maximum intensity of gas plasma glow to be observed. The
circular O-rings 32 help to provide a good seal with the
15 doors against the ingress of external air. The mixture of
reactive gas and aldehyde vapor is introduced in the process-
ing tunnel through a small pipe 33 with perforated holes 34.
The small pipe for gas and vapor introduction enters the
tunnel at one end and is positioned in the upper part of the
20 internal pipe 19 to allow uniform gas diffusion over the
entire tunnel length. In Figure 3, the RF inductive coil 35
is wrapped aroun~ the main external body of the processing
tunnel 17.
Figure 4 illustrates another embodiment of the in-
25 vention utilizing the microwave frequencies range from 100 MHz
to 300,0Q0 MHz~ The microwave gas plasma sterilizer shown in
Figure 4 consists of a metal housing 35 quite similar to those
used in conventional microwave ovens. Located within the
housing are the main components of the low temperature micro-
._ _ ,___,. ,,, , . , . . 1
~ 10432Z6
wave gas plasma sys-tem, comprising a magnetron 36 which, by
means of a transformer, rectifier, and magnetic field circuit
contained in power pack 37, converts the AC current from the
main power line 38 into microwave energy. The high power beam
5 o~ microwave energy, typically at 2450 MHz, is contained in
a wave guide 39 and directed against the blades 40 of a fan
41 which rotates at a slow RPM (revolutions per minute). The
fan reflects the power beam, bouncing it off the walls,
ceiling, back and bottom of the oven cavity 42. At the
10 bottom of the oven cavity 42, a pyrex glass plate 43 trans-
parent to microwaves is suspended approximately one inch above
the metal bottom of the processing cavity. The instruments
or material 44 to be surface sterilized are placed inside a
gas tight sealed container 45 which is positioned in the oven
15 cavity 42 and rests upon the glass plate 43. The container
45 may be constructed of any material which is transparent to
microwave energy, includlng polymeric materials, such as, poly-
propylene, polyethylene, polystyrene and Teflon (trademark),
carton board, paper or special glass composition. The
20 container 45 is of parallelepipedic shape with an upper lid
46 also made of microwave transparent material.
The lid 46 has two openings 47 and 4g, each with a
stopcock or valve 49 and 50 to allow the formation of the gas/
aldehyde vapors mixture in a partial vacuum atmosphere of
25 pressure between 0.1 and 10 mm of mercury. The container
45 contains two trays 51 which support the items 44 to be
sterilized, for example, the illustrated plastic bottles for
ophthalmological solutions. The trays 51 are generally per-
forated to allow a more uniform diffusion of the ionized gas
~, ~
3;~2 ~
l7
plasma. In the lower tray, a p]astic cup 52 is inscrted
which contains the aldehyde solution 53 to be evaporated.
Due to the thermal effect of microwaves, the aldehyde solu-
tion is gradually evaporated in the gas plasma when the micro- '
5 wave energy is switched on. The carrier gas to be ionized is
delivered to container 45 through opening 47 from a gas bottle
(not shown) into a pressure line 54 which includes a constant
flow valve 55, a pressure gauge 56, and, if desired, a flow-
meter. The low pressure vacuum needed to empty the loaded
10 container 45 is created through opening 48 by vacuum line 57,
which is connected to a trap 58 and to a vacuum pump 59.
A complete sterilizing cycle for the embodiment of
Figure 4 is as follows: filling the trays 51 with the equip-
ment to be decontaminated, introduction of the aldehyde solu-
15 tion cup 52, air elimination by vacuum activation, introductionof the carrier gas, and switching on microwaves during the
necessary time period, typically between 5 and 20 minutes,
to maintain a continuous plasma flow. At the end of the
exposure time, there is an automatic shut down of the
20 microwave generator 41, the carrier gas flow is also stopped
and the vacuum is broken through the two way valve 60. The
door of the microwave oven cavity 35 is then opened and the
container 45 is removed after disconnecting the flexible
tubings fastened to the stopcocks 49 and 50. The loaded con-
25 tainer 45 can be maintained sterile, by the rapidly closingof the stopcocks 49 and 50, until there is a need to remove
- the decontaminated equipment under aseptic conditions. An
entire sterilization cycle generally lasts between 10 and 30
minutes. At no time during processin~ does the surface
30 temperature approach 100C. No de-aeration of the
,, 110~32Z
decontaminated equipment is needed since the oxidiæing plasma
does not leave detectable traces of chemical on the treated
surfaces.
The semicontinuous sterilizing process described
5 above with respect to the equipment shown in Figures 2, 3, 3A
and 4 can be adapted to deliver sterile instruments inside
packages if the package is punctured by a small hole giving
access to the ionized and excited gas mixture. At the end
of the sterilization, the package can be removed under white
10 room conditions and a small sterile tape then applied to
cover and seal the small hole. The sealing tape can be
fastened manually or by an automatic machine.
The present invention can be applied to variable
flow rates of different gases at different temperatures or
15 multiple pressures. Further, the structural details of the
; described apparatuses, the dimensions and shapes of their
members, such as tunnel or cavity sizes, and their arrange-
ments, for example, introducing aldehyde vapors in microwave
field through evaporation or by a bubbler in the carrier gas
20 line, may be modified, and certain members may be replaced by
other equivalent means, for example, RF coils may be replaced
by capacitive plates and magnetrons may be replaced by klystrons
or amplitron tubes, without departing from the scope of the
invention.
The invention is illustrated by the following
Examples. In these Examples, the sporicidal data presented
was, in all instances, obtained according to the USDA (United
States Department of Agriculture) approved fumigant sporicidal
test method described in the Official Method of Analysis of
30 the Association of O~ficial Analytical Chemists (12th Ed.,
19 1~0~322
Nov. 1975).
Two types o~ highly resistant strains o~ the
following species: B. subtilis (ATCC 19659) and Cl. sporogenes
(ATCC 3584), were used in the experiments. The spores carriers
5 were silk suture loops (L) and porcelain cylinders (C) which
carried a dry ~ores load of 106 to 109 microorganisms. The
spores carriers were individually suspended from a thin
cotton thread attached to the gas pipe at the top of the pro-
cessing chamber.
There was also added at the bottom of the processing
chamber several spore test strips wrapped inside an 1/2 inch~hic~ surgical gauze. These control spore strips (American
Sterilizer Co. "SPORDI" (Trademark) were made of Bacillus
subtilis (globigii) and Bacillus stearothermophilus. The
15 subtilis strain was said to need a 60 minute exposure at 300F
for complete kill in dry heat while it required one hour and
forty-five minutes at 130F to be destroyed in the presence
of ethylene oxide gas concentration 600 mg. per liter, 50%
~elative humidity). In all the experiments, the vacuum
20 dried, acid resistant AOAC strains of B. subtilis and Cl.
sporogenes proved to be far more resistant than the SPORDI
spores and, for the sake o~ simplicity, the results of the
SPORDI strips are not given in the data tables in the
Examples.
25 Example 1
A series of experiments was conducted in a device
as illustrated in Figure 2. The carrier gases used to form
the plasma were pure oxygen, argon, and nitrogen. The
aldehyde vapors added to the carrier gas were produced in a
.
.. _,
-- 110~322
bubbler with solutions of the following aldehydes: formalin ~-
(8~ formaldehyde) acetaldehyde, glyoxal, malonaldehyde,
propionaldehyde, succinaldehyde, butyraldehyde, glutaralde-
hyde, 2-hydroxyadipaldehyde, crotonaldehyde, acrolein and
5 bcnzaldehyde~ The carrier gas flow rate was between ~0 cc.
and 100 cc. per minute at room temperature (about 20 to 25C).
The average internal pressure was 0.5 mm of mercury. The -
emission frequency was 13.56 MHz and the average power
density output in the plasma processing chamber was about
10 0.015 watts per cubic centimeter. The minimum amount of
aldehydes maintained in the continuous gas plasma flow was
about l~ mg per liter.
Table l below shows the results of experiments
assessing the influence of exposure time with the various low
15 temperature aldehyde seeded plasmas. Control experiments
consisted both of the gas alone (no aldehyde) and of a non-
oxidizing plasma (hydrogen gas) with formaldehyde or glutar-
a}dehyde vapors. For each type of sporulated bacteria on the
specific carrier (loop or cylinder), .en samples were used.
20 In the tables, the results are shown on a "pass" or "fail"
basis respectively indicated by the letter "P", which
denotes no growth in any of 10 samples, and "F", which
denotes 1 to lO samples having bacterial growth a~ter proper
culturing and heat shocking. For the sake of clarity, all
25 "fail" tests which preceeded the first "pass" tests were
omitted since it is obvious that shorteE exposure times
correspond to "fail" tests. As may be seen from the results
of Table l, contact times between lO and 30 minutes can pro-
vide satisfactory cidal action, the individual contact times
30 depending on the type of aldehyde vapor utilized.
,, ' ~
,: .
liO4322
21
~ 0~ . ~ ,, L~
'J ~ G C C f~ 4
_ ~ ~ ~ k~ ~ C LL C_ p~
! ~ C'~
_
' .~
ov ~_) C C c. a. ~ c I
;: ~,C~ --~ C~. G 1~ 1~ ~
IJ~ C " ~
~ _l ~
O~ C C ~ D
_ _~ C t~ . _
, _ __ .
C ~
,~ C
0 0 ~ C~ C ~ Q. ~ ~
t"1~ ~ G D_ ~ ~ ~ ~ Ll. U-
L~ ~ Cl. C~ . . .
- V) ~ ~
O _ ~ ~ C~ D~
Z _ ~ Cl ~ G t;.,
~ _ _ _ .
U~
~S C .rl . ,
O 'vl~ ' CL C~ ~ ~a~
~ ,1~ _1 ~ ~ G. L~ R-
L~ ~ ~ O .
~ ) C) ~
- - -
c ~ v~ ~
H ,~ C ,
1~ ~ h --~ ~ ~ c ~ c~ u. P.
!~ u~ D. ~ .
U~ l ~
Z O~ ~,) ~ .
, ~ ~ C ~ ~ _ . I
l ~0 E-~ .
. ~ ~ Cc~ , ~ ',
Lr~ ~ c c ~ e~. ~ , .
~ C ~ C ~ D. ~
l ~ ~ ~ C~ .
. _, . _ ~
' V~ .C 1
,u) ~
h V~ C~
.~O h C) ''O ~ C --~ 0
~ ' 0
_rl~ O--t~ ~ S O C ~ 1., X C~
Ql: ~ CC td tl~ X C r~ ~r1 h 1~ ~,, ~ O
ffl ~ c> c E V O O ~ U ~ V O ~
_l ~q ~ ~ b o ~-- o ~ 3 ~ )~ O c
3~ ~-- O ¢ `-- ~ h ~
110~322
E~ample 2
Utiliziny the same experimental conditions as those
of Example 1, except that the exposure time was maintained
around 15 minutes while the power output was increased
successively from 0.001 watts per cm of processing chamber
to 0.015 to 0.1 watts per cm3, a further series of experiments
was conducted.
AS may be seen from the results set forth in
Table II below, no killing was achieved at the lowest
power density, but excellent results were often obtained in
the 0.015 to 0.1 watts per cm range. These results indicate
the increased killing power which is attained by the addition
of aldehyde traces in the gas plasma. Oxygen appeared the
best carrier among the gases used in this series of experiments.
All"fail"tests which preceeded the first "pass" tests were
omitted from Table II since it is obvious that lower power
densities correspond to "fail" tests~
23 110~322
o '~ ~ C _ I ,V~ C r~ ~ C ~ ~ ~ ~ ~ L~ '
_~ o _~ ~ ~ C ~ _ C '` P, ~ s~.
Z _ ~.
o U _
~ V'u C C~. ~ D. ~
Z g .~ ~ ~ . ~ ~ .
~ ~ ~ ~ ~ L~ . ~ ~~ ~ ~ U.
V. U.
. .
V~ _
,
I O ~V ~ D
o o_~ C~ .
~ L~ ~ ~ ~ uA ~ ~ a. ~ u. c~.
~ U ~ U.
U.
! o ~
I ~ ,u .
e . ~
~ ~ o~ a. ~c~.
L~ ~ U~ D, a. ~ C~. eL t:~. C~ ~ O. ~ U~ 1-.
_ ~ _ _ __
. Vl .,
O ~ . ~ ~
~ o CO~ ~ ~ D.
1~:1 1~ 0 ~.) CL t~. &, tl. C~ . ~ ~ ~ 1~
~ ~ a. ~
~3 z ~ ~ .1
~- o .~ _
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I "U~ .~ ~ ~ ~ .
I ~ 1 ~1 ~ C~ G 1~. ~ ~ ~ ~ P~ ~ i~ . ~
.
. .
~ ___ _ 'C= ... _ ~_~ .`
~3 I ~b ~ r
, ~ - h ~ ~ C ~ ~ = h ~ ? U 1 = . ~ ~ .
o,, I
110~32Z
24
Example 3
In a further series of experiments, the aldehydes
were vaporized from a 2% active ingredients solution and this
corresponded roughly to a consumption of 15 cc. during a 15-
5 minute run. However, when sampling the gas plasma, the concen-
tration of aldehyde was found equal to 10 mg. per minute for
a flow rate of 100 cc./min. This aldehyde concentration in
the gas phase was roughly half the value to be expected from
the va~orized aldehyde solution, indicating that approximately
10 half the active aldehydes was deposited on the wall of the
processing chamber.
The concentrations of aldehyde recited in Table III
(below) are those observed in the gas plasma under normal
operating conditions. As may be seen from the results, at
15 the lower level of 0.1 mg./min., no increase in sporicidal
activity was observed with any of the three gases used in
the tests. At the 1 mg./min. level, there were inconsistent
results. At the 10 mg./min. level, most of the aldehydes
boosted the sporicidal efficacy of the gas plasma. At the
20 100 mg./min. level, all aldehydes showed an increased
spores killing over what was observed with the aldehydes
alone or with a non-oxidizing gas, such as, hydrogen loaded
with aldehydes.
,,
. . .
-` 110~3Z2
u~ 25
O co~ C. C. C. ~ ~D. C~. ~ L.
~ ~ C~ C. C~ .
O o ~ ~ c,~ ~ ~
Z ~ . .
~ _ ~
o o _ _ ,
~ o ,V, . , .
Z o ~ ~ ~ C~. ~~ U~
~ ~ U~ U~
O ~ U G C. C ~ C~ C~ C. ~ U. Q. D. L:.
C~ LL Q. 1~ C~
t~
_ ~1 .
_ O _ .
~ .
O ~ G ~ ~D~ C~' ~ . . .
O ~ t~ Q~ ' ' .
_ 0~ ' C L~ .
Z _
, ~ ~ _ _
$ _~
. _ _ ~) a. ~ ~ ~ ~
l o ~, ~ c~ ,.~ ~
! _t D ~ Q.. ~ D. ~ ~ t~. 1~ Cl~ t-, a. ~ r~
. 3 t~ C~
l -o~
l ~ - - l ~
I a~
O ~
O ~0~ D. ~ D~Q. ~U.
H _ O --1 Cl. ~ . U, t~.
H O O y Cl. ~ ~ 1~ t~ ~ C~. ~ ~ D.
H _ P~ t~ ~L C~. 1~ C~. C~ ~ e,. U. ~ ~ U~
1~3 Z .~ .
1~1 ~ - ~ .
. ~ X _ _ _
~ o .V~ . . ,
, o _ ~ a. ~ ~ ~ ~ ~
.~' I _ ~1 ~ ~ ~
l . O _O ~D. C~. n~ ~ ~ c~ D~ U~ ~ e~. tL
., I _ tS~ ~ c~L~c~
_ . ... ,.......... _
,~n . ~_.
~ V~ * ~
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~ ~ ~) ~ h
X ~ ~ ~n ~ ~ ~i~
~ ~ ~ ~ o~ O ~ ~ h o
~ o ~o a~ ~
. ~ ~ , ~ ~7
O--;~ O O ~ ~ nl t~l O C ~: h 2 ~ o
e ~ ~ ~ E ~ O O ~ o O nl
;~ C~ ~ '~ ~ _ O U ~ h O S: :Z
Y ~ - ' O U ~ O .
~11 ~ ~ U~ , ~_ .
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I
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26 110~a3ZZ
Example 4
Table IV (below) shows the results observed when
replacing a single aldehyde composition by a mixture of two
different aldehydes or by a mixed formula containing an
5 aldehyde with a non-aldehyde biocidal compound, i.e. phenol.
A mixed composition gave the same results as a single
aldehyde solution as long as the total content in aldehydes
remained the same in the two formulas. The presence of the
phenol did not affect the aldehyde efficacy as a sporicidal
10 booster agent in the gas plasma.
Not reported on Table IV are a number of experiments
conducted with various solutions of germicidal agents other
than phenols. While maintaining the same concentration of
aldehydes, there was added the following ingredients in equal
15 concentration: haloyen compounds, such as, chloroisocyanurates,
for example, trichloro-S-triazinetrione and iodophors, for
example, PVP-iodine complex; inorganic salts, for example,
selenium sulfide; an alcoholic solution of zinc undecylenate;
ammonium quaternaries, such as, cetyl-pyridinium chloride;
20 organo sulfurs, such as, methylenebisthiocyanate and nitrogen
compounds of fatty amines, such as N-alkyl trimethylene
diamine. In no case was there detected a synergistic effect
due to the presence of these agents in the vapor phase.
There was noted, however, a slight increase in activity
25 (additive effects) each time the plasma vaporization led to
the dissociation of the chemical salt with a release of a
halogen. The strong corrosive effect of ionized halogens was
also observed and this renders impractical the use of such
chemicals in a seeded low temperature plasma gas.
... .
, . . ... . .
27 110432Z
~ ~ t~ tl. C~. tl~ 1~
Z 0~, .
O t~_1 t` t~. tl. t. t~4 . ~
~ _ _ _ ,
I l Z
- e ~) t_ C~. t~ D.. CL _ _
O .
Z D~ .
O t~1 tb C~ t~ LL _ ._
_I~ t` CL CL C~, tL 12.,
:~ ~.
~i _ . ~ t` t~ t t~A _ ~A_ ,_
A ~) Cl. CL tA. t ti. t2,,
~3 Z h
1~ ~
~3. a.e~Lel.~- .
- . _. ~ _ .. _.. __
~ .C~ 'O . ~
C ~ h E~ "OA v~Ul
W E .~j V ~ ~ ~
. A ~ 0 i i ~ ~ . ¦.¦
~4 ~ _ ~ S ~
o ~ :~ t t, ~ ~ ~0
A , ~ U :~ ` e
.. . . _ . . . . . . .
''- - .
28 ~ 322
Example 5
I
A further set of experiments was conducted in the
apparatus of Figure 4. Since these experiments were conducted
at higher frequencies than is the case for Examples 1 to 4,
the microwave glow discharge was more uniform inside an
experimental polysulfone container. The gas plasma pressure
(2mm. of mercury) was slightly higher than in previous tests
because microwave discharges are more difficult to initiate
and to sustain at low pressures (- 1 mm. of mercury) than
DC or RF discharges.
Due to the higher longevity and efficacy of free
radicals and ionized species in a microwave gas plasma, the
contact time was reduced to 10 minutes. The plastic-polysulfone
container transparent to microwaves had the following dimen-
sions: 15 x 35 x 25 cms. (volume 16.37 liters). The averagedensity of the electromagnetic energy inside the resonant
cavity of about 0.02 watts/cc was tuned at the nominal fre-
quency of 2540 MHz (- 25 MHz). The gas flow rate was adjusted
between 900 cc. and 1000 cc. per minute which corresponded to
an average aldehyde content of 18 mg./min. in the plasma
phase. During the 10 minutes processing, around 18 cc. of
each aldehyde solution of 2~ concentration by weight was
evaporated. This corresponded also to roughly twice the
amount actually present for reaction in the gas plasma.
One may be seen from the results set forth in Table
V, an increase in sporicidal ef~icacy results from the seeding
of the small amount of aromatic, heterocyclic, saturated or
unsaturated aldehydes in the electromagnetic continuous gas
plasma discharge. When vaporizing furfural, the concentration
-` 110~a3ZZ .
29
of this chemical in the oxygen flow stream was 0.0018~ by
volume, since this chemical has a lower explosive limit in
air of 2.1~ by volume. The 2~ aqueous solution was main-
tained at all times during evaporation below the open cup
5 flash point of this aldehyde which is around 68C. Besides
benzaldehyde, other aromatic aldehydes, such as, thiophen-
aldehyde, and pyridine-2-aldehyde have qualitatively shown
the same behavior.
C 30 110~3ZZ
o
~ ~ '
Z ~ _1 r, C~ ~ 1~
O ~1 .
~_ V~
Z ~
~ ~ ~ C ~ ~ C ~ C~ ~ ~ ~ U~
~ _~ CL~Cn.~ ~
~ __
~ .
O ~ c ~. c c~ ~. a., Q. ~ ~
l Z D. ~ ~ ~
i ~ ~ .
I eS~ Ul
. c~. c ~ ~ ~ a. c~ . P CL Cl~
3c~ , u.,
. .A _ ._
~ O . ~~
h ~ G C~ . C 1~ . ~ 1~.
Z G ~ C C ~ Q. ~ G D~ .~ C~ G ~4 U~
O ~ -
~ ~ ^ C C~ . ~ C Cl. ~ ~: ~ 2. ~L
~ _~ ^ r, C~ ~
~:i .,
_ ,.......... . _ ~_ ~.
~ .
l ~ .~ ~ C~ ~ 0~ D. a ~ o
l ~ 0 ~0 .C --~ ~ ' .~ ~ ~ C -- C~ .
~ ~ a o ~ ~ ~
o ~ ~ I 0~ h_4 o ~ ~ i~ ~ .
C~ _ ~ E J O O C~ ~ ~i; .
G ~ ~ ~ ~ ~ O ~ v ~ _ h O t ~!I J~t
~ ~ ~ ~ 2 r r ~ ~ U ~ ~ t~
~:10~132Z
31
In summary of this disclosure, the present invention
provides a plasma gas sterilization method having substanti.al
advantages over prior gas phase sterilization procedures.
Modifi.cations are possible within the scope of this invention.
