GB2066076A

GB2066076A - Sterilizing with gas plasmas and aldehydes

Info

Publication number: GB2066076A
Application number: GB8000096A
Authority: GB
Original assignee: Biophysics Research and Consulting Corp
Current assignee: Biophysics Research and Consulting Corp
Priority date: 1980-01-02
Filing date: 1980-01-02
Publication date: 1981-07-08
Also published as: GB2066076B

Abstract

Sterilization of the surfaces of objects is achieved by placing the same in a continuous flow of a low temperature, low pressure gas plasma, containing small amounts of aromatic, heterocyclic, saturated or unsaturated aldehydes alone or mixtures thereof. The gas plasma is a partially ionized gas composed of ions, electrons and neutral species, which may be formed by electromagnetic discharges at subatmospheric pressure in the 1 to 300,000 Megahertz range, and corresponds to a minimum average spatial energy density of 0.001 watts per cubic centimeter. The gas plasma may also contain other vaporized cidal agents. Contrary to most gaseous sterilization procedures, the method is safe, allows quick handling of heat sensitive items, does not corrode equipment and does not leave toxic residues.

Description

SPECIFICATION Seeded gas plasma sterilization 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 microor ganisms 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, onlytwo 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 hospital, 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 high level of formaldehyde gas since CH2O is stable in high concentrations only at temperatures above 80"C 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 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 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 atmosphere with traces of hydrochloric acid, it can quickly produce at 70"C and 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 80"C in autoclaves. This method is said to kill most sporulated microorganisms at the concentration normally encountered in hospital practice while decreasing the aldehyde residue on instruments. It requires a time exposure of two hours with a formalin concentration of 8 gr. per cubic foot of autoclave. However, despite the long contact time and the relatively high temperature, 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 formaldehyde vapors, besides their toxicity and irritating characteristics, are difficult to handle at room temperatures and they do not provide a fast and reliable method to satisfactorily handle most of the hospital and industrial applications.

In the past two decades 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 30"C., 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.

Endotracheal tubes which are not properly aerated can cause tracheitis or tissue necrosis.

Besides the risks due to the toxicity of ETO residues, other accidents have been reported due to the explosive 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 CO2orfluorinated hydrocarbons 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 sterilant method which was capable of as fast a sporicidal action without any drawbacks from the toxicological or environmental viewpoint.

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 economical approach from the running and investment cost viewpoint 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 contacting the surface with a low temperature gas plasma containing at least 10 mg/l of an aldehyde under a subatmospheric pressure.

The term "Sterilization" as used herein refers to sporicidal action againstBaclllussubtll!s ATCC (American Type Culture Collection) 19659 and Clostridium sporogenes (ATCC 3584) because they are the resistant microorganisms used in the fumigantsterilant test according to the requirements of the AOAC (Official Method of Analysis of the Association of Official Analytical Chemists, 12th ed., Nov. 1975).

The destruction of these two resistant species of spores by the AOAC procedure automatically to the destruction of other less resistant microorganisms, such as, mycobacteria, non-lipid and small viruses, lipid and medium size viruses, and vegetative bac teria.

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 spore is surrounded by an exosporium which is a loose sac peculiarto some 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 peptidoglycan), (c) a plasma membrane, and (d) a core or spore protoplast.

The first line of resistance of the spore to exogenous agents consists of the proteinaceous outer coats which contain keratin-like proteins. The stability of keratin structures is due to frequent primary valence cross links (disulphide bonds) and secondaryvalence 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 resistans 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 of cidal molecules, excited atoms or radicals inside the microorganism protoplast.

To alterthe multilayered outer coats and thus allow further penetration and possible interactions in the critical cortex or protoplast regions, a very active agent must be chosen, and it has been found that an ionized gas plasma is an excellent vehicle to provide reactive atoms, free radicals, and molecules which will drastically alterthe protective layers of bacterial, fungi, and spores. The presence of small amounts of aldehyde vapors in the ionized low temperature non-oxidizing gas plasma, in accordance with this invention, leads to the destruction of sporulated and non-sporulated microorganisms.

In the present invention, the objects to be decomtaminated are exposed to a continuous flow of low temperature gas 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 lowtemperature gas plasma is formed by gaseous electric discharges. In an electrical discharge, free electrons gain energy from the imposed electric field and lose this energy through collisions with neutral gas molecules. The energytransfer process leads to the formation of a 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 charge carriers are approxi mately equal. The plasma used in the present inven tion 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 10 9 to 1012 per cm3.

Contrary to the conditions found in arcs or plasma jets, the electron and gas temperatures are very different due to the lack ofthermal equilibrium In a glow discharge, the electron temperature can be ten to a hundred times higher than the gas temperature.

The latter prnpertyisimportantwhen sterilizirrgthe surfaces of thermalrysensitive materials.

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 such as electrnns and photons. Irr 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 othernucleic acids. However, in the case of spores which can reach one millimeter in diameter, photon energy can be quickly dissipated 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, therefore, more efficacious when dealing with the smaller non-sporulated bacteria. In the case of high resistance spores, the photonic action may contri butt two partial alteration of the disulphide rich proteins coat and thus facilitate the diffusion of free redicals, atoms, or excited molecules inside the core region.

In the present invention, small amounts of vaporized aldehyde monomers and free radicals present in a low temperature 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 mechanisms 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 reactive epoxides and other intermediates and free radicals 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 groupsisthe penetration inside the cortex layer whose major component is the polymer murein (or peptidoglycan). Murein is a large, crosslinked, netlike molecule. A conjugated attack by atomic oxygen and aldehyde radicals on the polymer rapidly shakes and modifies the tight polymer structure 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 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 aspartic p-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 of their germinating capabilities. The 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 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 contains the heriditary charter, a repressed protein synthesizing system, the enzymes necessary to initiate the synthesis of new enzymes and structurai materials, and, presumably, reserves for the supply of energy intermediates. The modifications taking place in the outer coats, cortex and plasma 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.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 considerable reduction in spore killing time to be achieved over the 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 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 temperatures below 100"C. No severe corrosive or toxic residuals are observed when adding aldehydes to a gas plasma.

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, while the second method consists of a parallel plate or capacitive 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 vapor is constantly moving under the continuous suction of a vacuum pump. To excite gases and vapors in the processing area, the radiofrequency energy delivered by a generator is coupled through an inductive coil wrapped around the processing chamber or by means of capacitive discharge plates 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 impedance of the amplifier plate output circuit and the tank 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 generator are coupled directly through high impedance connectors. 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 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 inserted in the electronic circu it to monitor the difference between forward and reflected power.

Gas 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 upto 100 MHz.

The gas plasma may also be formed at higherfre- quencies in the microwave region, with frequencies ranging 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 longer life time than those formed at radio frequencies and they can persist downstream quite a distance into the glowless region. 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 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 needed to kill sporulated and nonsporulated bacteria.

The invention is described further, by way of illustration, with reference to Figures 2 to 4 of the accompanying drawings, wherein: Figure 2 is a schematic representation of an apparatus for sterilizing various hospital type disposals in a semi-continuous manner; Figures 3 and 3A are sectional views of the sterilizing chamberof Figure 2; and Figure 4 is a schematic representation of an alternative 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 manner various hospital type disposals. The system comprises a tunnei-like processing chamber 1, having a door 2 at each end, only the door2 at the left-hand entrance side being shown. The disposables or non-disposables, for instance, plastic bottles of parenteral or ophthalmological solutions, 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 automati caily by means of an electrically driven mechanical system 3.The loaded tunnel processing chamber 1 is then subjected 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.

The gas to be ionized then is delivered from a compressed 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 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 numerous small holes properly spaced for an even distrubtion 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 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 powerwattmeter 14 and an RF generator 15 converting AC (alternating current) standard current into 13.56 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 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 type of installation, a single inductive coil extending over the entire tunnel length may be driven from a single power generator or a series of smaller coil sections may be operated from smaller modular type RF generators.

During RF excitation, continuous removal of gas 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 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 1 is then ready for sterilizing a new load. The entire sterilization cycle time generally takes between 10 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, as shown in Figure 2. The tunnel 17 is of cylindrical shape around a main axis and essentially consists of two concentric cylindrical pipes 18 and 59 made of highly resistant inert material, such as, glass or a polymeric material, for example, a polysulfone, which are held by compression on end flanges with silicone type O-rings 20. After assembling the internal pipe 19 inside the external pipe 18, a hollow space ring 21 is created in which vacuum and subatmospheric pressure is provided by vacuum pump suction through bottom openings 22. To permit a subatmospheric atmosphere to be formed around the objects to be decontaminated, 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 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 180 around the hinge 31. The front and end doors of the tunnel are generally made of a dark ultraviolet absorbing 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 doors against the ingress of external air.The mixture of reactive gas and aldehyde vapor is introduced in the processing 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 internal pipe 19 to allow uniform gas diffusion over the entire tunnel length. In Figure 3, the RF inductive coil 35 is wrapped around the main external body of the processing tunnel 17.

Figure 4 illustrates another embodiment of the invention utilizing the microwave frequencies range from 100 MHzto 300,000 MHz. The microwave gas plasma sterilizer shown in Figure 4 consists of a metal housing 35 quite similar two those used in conventional microwave ovens. Located within the housing are the main components of the low temperature microwave gas plasma system, 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 of 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 bottom of the oven cavity 42, a pyrex glass plate 43 transparent 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 cavity 42 and rests upon the glass plate 43. The container 45 may be constructed of any material which is transparent to microwave energy, including polymeric materials, such as, polypropylene, polyethylene, polystyrene and Teflon (trademark), carton board, paper or special glass composition. The container 45 is of parallelepipedic shape with an upper lid 46 also made of mocrowave transparent material.The lid 46 has two openings 47 and 48, 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 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 opthalmological solutions. The trays 51 are generally perforated to allow a more uniform diffusion of the ionized gas plasma. In the lower tray, a plastic cup 52 is inserted which contains the aldehyde solution 53 to be evaporated. Due to the thermal effect of microwaves, the aldehyde solution is gradually evaporated in the gas plasma when the microwave 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 flowmeter. The low pressure vacuum needed to empty the loaded 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 equipment to be decontaminated, introduction of the aldehyde solution cup 52, air elimination by vacuum activation, introduction of the carrier gas, and switching on microwaves during the necessary time period, typically between 5 and 20 minutes, to main tain a continuous plasma flow. At the end of the exposure time, there is an automatic shut down of the 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 romeved after disconnecting the flexible tubings fastened to the stopcocks 49 and 50.The loaded container 45 can be maintained sterile, by the rapidly closing of 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 processing does the surface temperature approach 100"C. No de-aeration of the decontaminated equipment is needed since the oxidizing plasma does not leave detectable traces of chemical on the treated surfaces.

The semicontinuous sterilizing process described 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 ofthe sterilization, the package can be removed under white 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 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 arrangements, for example, introducing aldehyde vapors in microwave field through evaporation or by a bubbler in the carrier gas 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 the Association of Official Analytical Chemists (12th Ed., Nov. 1975).

Two types of highly resistant strains of the following species: B. subtilis (ATCC 19659) and Cl.

sporogenes (ATCC 3584), were used in the experiments. The spores carriers were silk suture loops (L) and porcelain cylinders (C) which carried a dry spores load of 106 to 109 9 microorganisms. The spores carriers were individually suspended from a thin cotton thread attached to the gas pipe at the top of the processing chamber.

There was also added at the bottom of the processing chamber several spore test strips wrapped inside an 1/2 inch thick surgical gauze. These control spore strips (American SterilizerCo. "SPORDI" (Trademark) were made of Bacillus subtilis (globigii) and Bacillus stearothermophilus. The subtilis strain was said to need a 60 minute exposure at 300 F for complete kill in dry heat while it required one hour and forty-five minutes at 130'to be destroyed in the presence of ethylene oxide gas concentration 600 mg, per liter, 50% relative humidity).In all the experiments, the vacuum 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 of simplicity, the results of the SPORDI "strips are not given in the data tables in the Examples.

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 nit rogen. The aldehyde vapors added to the carrier gas were produced in a bubbler with solutions of the following aldehydes: formalin (8% formaldehyde) acetaldehyde, glyoxal, malonaldehyde, propional dehyde, succinaldehyde, butyraldehyde, glutaral dehyde, 2- hydroxyadipaldehyde, crotonaldehyde, acrolein and benzaldehyde. The carrier gas flow rate was between 80 cc. and 100 cc. per minute at room temperature (about 20 to 25 C). The average inter nal 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 0.015 watts per cubic centimeter.The minimum amount of aldehydes maintained in the continuous gas plasma flow was about 10 mg per liter.

Table 1 below shows the results of experiments assessing the influence of exposure time with the various low 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 glutaraldehyde vapors. For each type of sporulated bacteria on the specific carrier (loop or cylinder), ten samples were used. 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 10 samples having bacterial growth after proper culturing and heat shocking. For the sake of clarity, all "fail" tests which preceeded the first "pass" tests were omitted since it is obvious that shorter exposure times correspond to "fail" tests. As may be seen from the results of Table 1, contact times between 10 and 30 minutes can provide satisfactory cidal action, the individual contact times depending on the type of aldehyde vapor utilized.

TABLE I

CARRIER GASES OXYGEN ARGON NITROGEN Exposure Time (min) 10 15 30 10 15 30 10 15 30 10 15 30 10 15 30 10 15 30 Type of Vaporised Aldehydes B. subtilis Cl. sporogenes B. subtilis Cl. sporogenes B. subtilis Cl. sporo.

in Carrier Gas LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC Formaldehyde PP PP PP PP PP PP Acetaldehyde PP FP PP PP FP PP FP PP FP PP Glyoxal PP PP PP PP PP FP PP Malonaldehyde PP PP PP PP PP PP Propionaldehyde PP PP PP FP PP PP FP PP Succinaldehyde PP FP PP PP FP PP FP PP FP PP Butyraldehyde PP PP PP PP PP FP PP Glutaraldehyde PP PP PP FP PP PP FP PP 2 Hydroxyadipaldehyde PP PP PP PP PP PP Acrolein PP PP PP PP PP PP Crotonaldehyde PP PP PP PP PP PP Benzaldehyde PP PP PP PP PP FP (Hydrogen- ) CONTROLS (Formaldehyde) FP FF FF FF FF FF Carrier Gas Alone PP FP PP FP FP PP (no aldehyde) Example 2 Utilizing 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 cm3 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 cm3 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.

TABLE II

CARRIER CASES OXYGEN ARGON NITROGEN Power Density 1 15 100 1 15 100 1 15 100 1 15 100 1 15 100 1 15 100 (10-3 watts/cc) Type of vaporised aldehydes B. subtilis Cl.sporogenes B.subtilis Cl.sporogenes B.subtilis Cl.sporogenes in carrier gas LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC Formaldehyde PP PP PP PP PP PP Acetaldehyde PP FP PP FP PP FP PP FP PP Glyoxal PP PP PP PP PP PP Malonaldehyde FF PP FF PP FF PP FF PP FF PP FF PP Propionaldehyde PP PP PP FP PP PP FP PP Succinaldehyde PP PP PP PP PP PP Butyraldehyde PP PP PP PP PP FP PP Glutaraldehyde PP PP PP PP PP PP 2 Hydroxyadipaldehyde FF PP FF PP FF PP FF PP FF PP FF PP Acrolein PP PP PP PP PP PP Crotonaldehyde PP FF PP PP FF PP PP FF PP Benzaldehyde FF PP FF PP FF PP FF PP FF PP FF PP (Hydrogen CONTROLS (Formaldehyde FP FF FF FF FF FF Carrier Gas Alone FF PP FP FF PP FF FP FF FP FF FP no aldehyde) Example 3 In a further series of experiments, the aldehydes were vaporized from 2% active ingredients solution and this corresponded roughly to a consumption of 15 cc. during a 15-minute run. However, when sampling the gas plasma, the concentration 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 vaporized aldehyde solution, indicating that approximately half the active aidehydes was deposited on the wall of the processing chamber.

The concentrations of aldehyde recited in Table Ill (below) are those observed in the gas plasma under normal operating conditions. As may be seen from the results, at 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 efficac/ of the gas plasma. At the 100 mg./min. level, all aldehydes showed an increased spores killing over when was observed with the aldehydes alone or with a non-oxidizing gas, such as, hydrogen loaded with aldehydes.

TABLE III

CARRIER GASES OXYGEN ARGON NITROGEN Vaporised Aldehydes 0.1 10 100 0.1 10 100 0.1 10 100 0.1 10 100 0.1 10 100 0.1 10 100 Flow Rate (mg/mm) Type of vaporised Aldehydes B.subtilis Cl.sporogenes B.subtilis Cl.sporogenes B.subtilis Cl.sporogenes in carrier gas LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC Formaldehyde PP PP PP PP PP PP Acetaldehyde PP FP PP FP PP FP PP FP PP Glyoxal PP PP PP PP PP PP Malonaldehyde FF PP FF PP FF PP FF PP FF PP FF PP Propionaldehyde PP PP PP FP PP PP FP PP Succinaldehyde PP PP PP PP PP PP Butyraldehyde PP PP PP PP PP FP PP Glutaraldehyde PP PP PP PP PP PP 2-Hydroxyadipaidehyde FF PP FF PP FF PP FF PP FF PP FF PP Acrolein PP PP PP PP PP PP Crotoneldehyde PP FF PP PP FF PP PP FF PP Benzaldehyde FF PP FF PP FF PP FF PP FF PP FF PP (Hydrogen-Glutar CONTROLS(aldehyde FF FF FF FF FF FF Carrier Gas Alone FF FF FF FF FF FF (no aldehyde) 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 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 booster agent in the gas plasma.

Not reported on Table IV are a number of experi- ments 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 concentration; halogen compounds, such as, chloroisocyanurates, for example, trichloro - S - triazinetrione and idophors, for example, PVP-iodine complex; inorganic salts, for example, selenium sulfide; an alcoholic solution of zinc undecylenate; ammonium quaternaries, such as, cetyl-pyridinium chloride; organo sulfurs, such as, methylenebis thiocyanate 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 (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.

TABLE IV

CARRIER GASES OXYGEN ARGON NITROGEN Type of aldehydes mixture B.subtilis Cl.sporogenes B.subtilis Cl.sporogenes B.subtilis Cl.sporogenes (2% total content in aldehydes) L C L C L C L C L C L C Formaldehyde + Glutaraidehyde P P P P P P P P P P P P Succinaldehyde + Formaldehyde P P P P P P P P P P P P Glutaraldehyde + Phenol P P P P P P P P P P P P Butyraldehyde + Glutaraldehyde P P P P P P P P P P P P Formaldehyde + Acetaldehyde P P P P P P P P P P P P CONTROLS Carrier Gas Alone F F F F F F F F F F F F (no aldehydes) Example 5 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 dimensions: 15 x 35 x 25 cms. (volume 16.37 liters). The average density of the electromagnetic energy inside the resonantcavity of about 0.02 watts/cc was tuned at the nominal frequency 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 efficacy 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 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 maintained at all times during evaporation below the open cup flash point of this aldehyde which is around 68"C. Besides benzaldehyde, other aromatic aldehydes, such as, thiophenaldehyde, and pyridine - 2 - aldehyde have qualitatively shown the same behavior.

TABLE V

CARRIER GASES OXYGEN ARGON NITROGEN Type of Vaporized B.subtilis Cl.sporogenes B.subtilis Cl.sporogenes B.subtilis Cl.sporogenes Aldehydes LC LC LC LC LC LC in carrier gas Formaldehyde PP PP PP PP PP PP Acetaldehyde PP PP PP PP PP PP Glyoxal PP PP PP PP PP PP Malonaldehyde PP PP PP PP PP PP Propionaldehyde PP PP PP PP PP PP Succinaldehyde PP PP PP PP PP PP Butyraldehyde PP PP PP PP PP PP Glutaraldehyde PP PP PP PP PP PP 2-Hydroxyadipaldehyde PP PP PP PP PP PP Acrolein PP PP PP PP PP PP Crotonaldehyde PP PP PP PP PP PP Benzaldehyde PP PP PP PP PP PP Furfural PP PP PP PP PP PP CONTROLS Carrier Gas FP FF FP FF FF FF Alone (no aldehydes) In summary of this disclosure, the present invention provides a plasma gas sterilization method having substantial advantages over prior gas phase sterilization procedures. Modifications are possible within the scope of this invention.

Claims

1. A method of sterilizing a surface comprising contacting the surface with a low temperature gas plasma containing at least 10 mg/l of an aldehyde under subatmospheric pressure.

2. A method as claimed in claim 1 in which the aldehyde is an aromatic, heterocyclic, or saturated acyclic or unsaturated acyclic aldehyde.

3. A method as claimed in claim 1 or 2, in which the aldehyde is formaldehyde, acetaldehyde, glyoxal, malonaldehyde, propionaldehyde, succinaldehyde, butyraldehyde, glutaraldehyde, 2 - hydroxyadipaldehyde, acrolein, crotonaldehyde, benzaldehyde or furfural.

4. A method as claimed in any one of claims 1 to 3, in which the pressure of the gas plasma is 0.1 to 10 mm of mercury.

5. A method as claimed in claim 1 or 2 in which the gas plasma is produced by electromagnetic excitation of oxygen, argon, helium, nitrogen, carbon dioxide, nitrogen oxide or a mixture of two or more of such gases.

6. A method as claimed in any one of claims 1 to 5 in which the gas plasma is produced by electromagnetic discharges in the 1 to 100 MHz radio frequency region.

7. A method as claimed in any one of claims 1 to 5 in which the gas plasma is produced by gaseous electromagnetic discharges in the 100 to 300,000 MHz microwave range.

8. A method as claimed In claim 6 or 7 in which the gas plasma is confined inside a fluid-tight chamber and the electromagnetic field density in the chamber is at least 0.001 watts per cubic centimeter.

9. A method as claimed in any one of claims 1 to 8, in which the aldehyde is introduced to a continuously-produced gas plasma stream in a carrier gas flow upstream of the sterilization location.

10. A method as claimed in any one of claims 1 to 8 in which the aldehyde is introduced to a continuously-produced gas plasma stream in a plasma forming chamber in which the sterilization is also effected.

11. A method as claimed in any one of claims 1 to 10 in which the gas plasma also contains a vaporized biocidal agent.

12. A method as claimed in claim 11, in which the biocidal agent is phenol, halogen, inorganic or organic metal salt, an organosulfur or nitrogen compound, or a mixture of two or more of such agents.

13. A method as claimed in claim 11 or 12, in which the biocidal agent is present in the gas plasma in an amount of at least 10 mg/l.

14. A method of sterilizing a surface substantially as hereinbefore described with reference to any one ofthe Examples.

15. A method of sterilizing a surface substantially as hereinbefore described with reference to, and as illustrated in, Figures 2 to 3 of the accompanying drawings.

16. A method of sterilizing a surface substantially as hereinbefore described with reference to, and as illustrated in, Figure 4 of the accompanying drawings.

17. Asterile surface whenever produced buy a method as claimed in any one of claims 1 to 16.