Disinfection was investigated in high-density ozone produced by dielectric barrier discharge under atmospheric pressure. Disinfection was studied on three-dimensional carriers made of hydrophilic vinyl-polysiloxane impression material and granular acrylic resin used for oral surgery. Experimental results indicate disinfection of spore-forming bacteria: Geobacillus stearothermophilus and Bacillus atrophaeus, and selected species of opportunistic pathogens: Aspergillus niger, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Candida albicans.
Advanced scheme for the sterilization and high-grade disinfections of patient-care products has been drawn attention increasingly. Increasing consideration has been required in medical institutions for nosocomial infection in patients, cross-infection among medical facilities staff, and secondary infection of immune deficient patients. This is not only due to emerging diseases such as enterohemorrhagic Escherichia coli (EHECO-157), human immunodeficiency virus (HIV), severe acute respiratory syndrome virus (SARS), and Ebora hemorrhagic fever but also due to the increase in infection by normal flora in the soil and human skin, such as Acinetobacter, Candida albicans, Staphylococcus aureus etc. Advanced schemes for the sterilization of medical-care materials are being increasingly drawn attention. Because of the possibility of infection by blood-originating pathogens, certain precautions are needed for handling medical-care materials, such as dental impression, as an integral part of dental practice of infection control for dental health-care personnel [
Infection control, using disposable patient-care products such as gloves, has certain limits. The application of on-site disinfection using certain surgery equipment and materials can reduce the risk of infection in medical treatment. In this report, we describe the study of a disinfection system using high-density ozone produced by atmospheric-pressure dielectric barrier discharges. Conventional methods for preventing infection are widely accepted. Disinfection using high-pressure steam in an autoclave, however, can introduce surface damage to metallic surgical tools and distortion of delicate plastic parts of medical-care products. For disinfection using liquid chemical agents, certain species of pathogenic bacteria acquires resistance and infection by a multiresistant pathogen is a serious biohazard. Some chemical disinfectants are also toxic. Although gas sterilization using ethyleneoxide gas (EtO) plays a primary role in low-temperature sterilization, treatment using EtO should meet rigorous protocols, because EtO is an agent that reacts with many constituents of tissue resulting in cellular and tissue dysfunctions and damages. Human carcinogenicity by EtO is a strong concern. The United States Department of Health and Human Services (DHHS) has determined that EtO may reasonably be anticipated to be a human carcinogen [
Storage of such chemical compounds is facing increased costs and the risk of leakage in accident fears. The history of ozone disinfection begins in the 1890s. Disinfection using water-diluted ozone is a popular and reliable method for cleaning. Recent progress in ozone generation technique has enabled production of ozone in quantities as high as 50,000 to 100,000 ppm, in the semiconductor industry mask-removing process. Oxidation by high-density ozone can disinfect spore-forming bacteria. To assess the antibacterial effect, the mortality of microorganisms was examined using spore-forming bacteria: Bacillus atrophaeus (formerly Bacillus subtilis var niger) and Geobacillus stearothermophilus (formerly Bacillus stearothermophilus); a dental yeast-like fungus, Candida albicans and selected species of pathogenic bacteria, Aspergillus niger, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus [
The wall temperature was maintained at constant value with PID controlled flexible heaters to maintain the temperature in the internal space at 50˚C. Biological indicators were located at the center of the reactor, at 140 mm from the bottom and each corner inside. The reactor chamber dimensions were 31 L, 360 mm wide, 270
Ozone disinfection reactor and build up of ozone density
mm deep, and 320 mm high, and made from aluminum with no artificially anodized coating. Steam from distilled water, 0.5 mL∙min−1 (liquid phase), was generated in a perforated brass block, heated to 120˚C and supplied into the reactor chamber. The relative humidity in the reactor chamber was maintained at 72%. The value was determined by the condensation at the lowest temperature from the chamber wall. Ozone mixture working gas was decomposed by using a manganese peroxide-coated carbon honeycomb catalysis, and the outflow to the exterior environment was controlled to be lower than 0.05 ppm. This emission level meets the operational exposure limits in the United Kingdom, other parts of Europe, the United States, Canada, and Japan: 0.1 ppm for regular exposure. The ozone density in the exhaust gas was measured with a GASTEC absorption tube. Deterioration of plastic material was measured with IR absorption using a microscopic Fourier-Transform Infrared (FTIR) spectrometer (Jansen Microscopic-FTIR) and processed with a workstation (WS/IR-8300, Jasco Co.). Attenuated total-reflectance (ATR) data are not available for these samples.
In the first experiment, a biological indicator, made of Schleicher & Schuell filter paper #470, 6.4 × 38.1 mm in width and length, respectively, was used as a carrier for Bacillus atrophaeus ATCC 9372 spores of 2.0 × 106 CFU. ATCC stands for the American Type Culture Collection. CFU stands for colony-forming units, the density of active end-spores to be detected as individual colonies after incubation. Samples of microorganisms were selected from the library of the Raven Biological Laboratories Company.
Although maximum disinfection grade can be obtained with direct exposure, radicals are supplied through a small perforation of a porous sheet covering a plastic sterile casing. Thus, the compatibility study with such sterile packaging is important for use in transportation and for storage. Compatibility involves two conditions: #1. The antibacterial effect is sufficient in the region separated by the porous sheet; #2. The disinfection process will not deteriorate the sheet or the sterile casing. The selected package materials were polyethylene, poly(propylene), glassine paper, and a Tyvek sheet. Glassine paper is commercially available wax-applied cellophane, with small holes approximately of 1 μm in size at a density of 200 holes per 1 mm2. Tyvek is a high-density polyethylene non-woven fabric of 0.15 mm in thickness. The density of microscopic apertures in this non-woven fabric is not identified. The selected microorganisms were spore-forming, gram-positive bacteria: Geobacillus stearothermophilus ATCC 7953, Bacillus atrophaeus ATCC 9372, and Bacillus subtilis ATCC 6653. G. stearothermophilus is approved as a standard indicator for autoclaving, which is pressurized, high-temperature steam sterilization, and B. atrophaeus is an approved indicator for gas sterilization using ethylene oxide. From a clinical view, opportunistic pathogens: Aspergillus niger ATCC 16404, Pseudomonas aeruginosa ATCC 9027, Escherichia coli JCM 1649 Staphylococcus aureus ATCC 6538, and a yeast-like fungus, Candida albicans ATCC 10231, were prepared. JCM stands for the Japan Collection of Microorganisms, a library of available microorganisms. The density of the microorganisms was adjusted in the following way. Selected species were incubated in a soybean casein digest (SCD)—fluid culture medium. A small amount of culture fluid was transferred to an SCD-agar culture medium and incubated. A small amount of the cultured sample was diluted in distilled water, and density was adjusted to approximately 106 CFU per 0.1 mL. An amount of 0.1 mL of the suspension was applied to sterilized carriers and formed circular region of 8 to 10 mm in diameter after drying. The carrier materials were a stainless steel plate of 15 × 15 mm2 and 1.5 mm in thickness, a slide glass, a filter paper carrier, and a dental impression made of VPS. The hydrophilic surface characteristics and flexibility of VPS allow stronger attachment of microorganisms, compared with a smooth surface, such as a slide glass. The selected material is commercially available Exafine from GC Medical Corp., Japan. This material is equivalent to Ewaflex, marketed by GC America in the United States, Exafine samples were cut out into 15 × 15 mm2 samples, including one or two impressions of teeth, Candida albicans ATCC 10231 (2.2 ×106 CFU) was applied onto the carriers. Reference biological indicators were prepared with Candida albicans applied on a stainless plate, and the sterilization time was compared. After the disinfection process, the biological indicator was incubated in 100 mL of soybean casein digest (SCD)—culture fluid medium. In this process, we followed protocols of Japanese Pharmacopeias [
The final experiment involved the disinfection of dental adhesive resin contaminated with Aspergillus niger and Candida albicans. The selected material was Superbond C & B from SunMedical Co. in Japan. This compound is equivalent to C & B-Metabondfrom Porkell Inc., marketed in North America. Super bond C & B is a self- curing dental adhesive system containing granular poly (methyl methacrylate) (PMMA) resin and a liquid: 5%
. Variation of sterilization efficiency: dependence on packaging materials
Material | Temperature | Thickness | Exposure time (min) | |||
---|---|---|---|---|---|---|
(mm) | 30 | 60 | 90 | 120 | ||
Glassine | 30 | 0.05 | + | + | + | − |
50 | 0.05 | + | + | + | − | |
70 | 0.05 | + | + | − | ||
Tyvek | 30 | 0.15 | − | − | − | |
50 | 0.15 | + | − | − | − | |
70 | 0.15 | + | − | − |
Biological indicator: B. atrophaeus ATCC 9372 (2.0 × 106 CFU).
. Sterilization of spore forming bacteria in Tyvek package
Selected species and populations | Exposure time (min) | |||
---|---|---|---|---|
30 | 60 | 90 | 120 | |
G. stearothermophilus ATCC 7953 (1.76 × 105 CFU) | + | + | − | |
B. atrophaeus ATCC 9372 (8.48 × 105 CFU) | + | + | − | |
B. subtilis ATCC 6633 (1.58 × 106 CFU) | + | + | − |
. Variation of sterilization efficiency: dependence on carrier material
Carrier materialb | Result |
---|---|
Stainless steel (SUS304) | + + |
Pyrex glass (0.18 mm) | − − |
Filter paper | − − |
bBiological indicator: B. atrophaeus ATCC 9372 (8.48 × 105 CFU).
(a)
(a)
3 M Attest R 1264/1262 | Humidity | Exposure time (min) | |||
---|---|---|---|---|---|
At 50˚C | 15 | 20 | 25 | 30 | |
B. atrophaeus (AttestTM 1264) | 100% | + | + | + | − |
G. stearothermophilus (AttestTM 1262) | 100% | + | + | − | − |
(b)
(b)
Biological indicator | Package type | Exposure time (min) | |
---|---|---|---|
At 50˚C | 30 | 45 | |
B. atrophaeus ATCC 9372 (1.0 × 106 CFU) | Tyvek package | − | − |
Glassine | −c | −c | |
G. stearothermophilus ATCC 7953 (1.3 × 106 CFU) | Tyvek package | − |
cDamage of bonding observed.
(c)
(c)
Biological indicator | Temperature | Exposure time (min) | ||
---|---|---|---|---|
˚C | 5 | 10 | 15 | |
B. atrophaeus ATCC 9372 (1.0 × 106 CFU) | 50 | + | − | − |
65 | − | − −d |
dDamage of non-woven sheet observed.
. Disinfection of opportunistic pathogen in Tyvek package
Biological indicator and population | Exposure time (min) | ||
---|---|---|---|
10 | 20 | 30 | |
Aspergillus niger ATCC 16404 (7.83 × 105 CFU) | − | − | − |
Pseudomonas aeruginosa ATCC 9027 (3.36 × 106 CFU) | − | − | − |
Escherichia coli JCM1649 (1 × 106 CFU) | − | − | − |
Staphylococcus aureus ATCC 6538 (5.48 × 106 CFU) | − | − | − |
Candida albicans ATCC 10231 (1.32 × 106 CFU) | − | − | − |
4-META/95% MMA-TBB. MMA is methyl methacrylate monomer, 4-META 4-methacryloxyethyl trimellitate anhydride, and TBB: tributyl-borane hardening catalyst. This material is applied in oral surgery as a coating and teeth adhesive (enamel and dentin), metallic parts and dental resin.
The experimental result is summarized as following tables. In the tables “−” minus indicates the successful disinfection and “+” plus indicates a denial of sterility.
In the next experiment, higher density was realized using a small reactor made of acrylic-Lucite with 40 mm internal diameter.
Disinfection of opportunistic pathogen in Tyvek package. (a) Aspergilles niger ATCC 16404 (7.83 × 105 CF); (b) Popsuedomonas aeruginosa ATCC 9027 (3.36 × 106 CFU); (c) Escherichia coli JCM1649 (1 × 106 CFU); (d) Sta- phylococcus aureus ATCC 6538 (5.48 × 106 CFU); (e) Candida albicans ATCC 10231 (1.32 × 106 CFU)
Exafine VPS tooth impression and acryl resin for filler
Disinfection of Candida albicans applied to Exafine
Disinfection of Candida albicans in ozone reactor: location of biological indicator A to J
. Disinfection of Candida albocans applied to Exafine impression material
Carrier and population | Exposure time (min) | ||||
---|---|---|---|---|---|
5 | 10 | 15 | 20 | 30 | |
Stainless steel (1.32 × 106 CFU) | - | - | - | ||
Exafine (1.43 × 106 CFU) | + | + | - | - |
Biological indicator: Candida albicans ATCC 10231.
. Disinfection of Candida albicans applied to Exafine impression material in an ozone reactor
Exposure time (min) | Position of indicatora | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
A | B | C | D | E | F | G | H | I | J | |
15 | - | - | - | - | - | - | - | - | - | - |
30 | - | - | - | - | - | - | - | - | - | - |
aBiological indicator: Candida albicans ATCC 10231 (1.4 × 106 CFU).
Next, we show the disinfection of Aspergillus niger in granular acrylic resin. A PMMA resin of 0.16 g was measured with a small-sized measuring spoon and placed in a small petridish, and a distilled water suspension of Aspergillus niger was applied. The final population was adjusted to be approximately to 3.4 × 105 CFU. Similar experiment was repeated for Candida albicans (1.7 × 106 CFU) sample. The top of acryl resin shows more like solid shell than granular resin. After the disinfection cycle, whole resin samples were incubated in an SCD fluid culture medium. The incubation result shows that disinfection was completed in 10 and 15 minutes, without significant deterioration in solidity when mixed PMMA resin was formed. The results are summarized in
. The sterilization time was compared for Candida albicans, in a series of plain and shaped Exafine carrier and composite Exafine carrier with narrow gaps. The following bactericidal effect was observed inside sterile packages of a Tyvek. Although opportunistic pathogenic bacteria were sterilized within short time, inactivation of spore-forming bacteria of Bacillus genera required longer time. Experimental results indicate that mixing with water vapor is essential. This result suggests speculation for the mechanism of disinfection based on hydroxyl radicals generated in reactions
Production of hydroxyl radical:
Vapor injection plays an essential part; injection of dry ozone was tested and was shown to require an operational time for sterilization of twice that or longer than the values measured here. Low-temperature vapor itself is also ineffective. The measured incompatibility in package materials of polyethylene and poly (propylene) indicates that discretion is needed in the package material selection. A porous sheet must be used for ozone disinfection.
A grading system set forth by Ozonesolutions Co. [
. Disinfection of granular acrylic resin contaminated
Biological indicator and population | Exposure time (min) | |
---|---|---|
10 | 15 | |
Aspergillus niger ATCC 16404 (3.4 × 105 CFU) | - | - |
Candida albicans ATCC 10231 (1.7 × 106 CFU) | - | - |
. Antibacterial effect
Microorganism | ||
---|---|---|
Gram-stain positive | Gram-stain negative | Yeast/Fungus |
S. aureus | E. coli, P. aeruginosa | C. albican, A. niger |
Disinfected | Disinfected | Disinfected |
FTIR for control and treated samples. (a) FTIR of untreated control; (b) FTIR of treated material
. Conformity grade of solid materials
Conformity Grade | Material |
---|---|
(Alphabetical Order) | |
A Excellent | Butyl, Chemraz (Perfluoro Elastomer), CPVC (Chlorinated Polyvinyl Chloride), Cross-Linked Polyethylene (PEX), Durachlor-51, EPR, Ethylene-Propylene, Vinyl-Polysiloxane Exafine, Fluorosilicone, Glass, Hastelloy-C, HDPE (High-Density Polyethylene), Inconel, Kalrez, Kel-F, (PCTFE), Kynar, Millable (Polyurethane Rubber, Heat Cured Rubber, Heat Vulcanized Rubber), PEEK (Polyether Ether Ketone), Polycarbonate, Poly(Methyl Methacrylate) (PMMA) Resin, PVC, PVDF (Polyviniliden Fluoride), Santoprene (Thermoplastic Vulcanizates), Silicone, Stainless Steel 304/316, Teflon (Polytetrafluoroethylene), Titanium, Vamac (Ethylene Acrylic Elastomer), Viton (Fluoropolymer Elastomer) |
B Good | ABS Plastic, Aluminum (Dry Ozone), Brass, Bronze, Copper, EPDM (Ethylene Propylene Diene Monomer Rubber), Flexelene (Polyolefin), LDPE (Low-Density Polyethylene), Polyacrylate, Polyethelyne, Polysulfide, Polyacrylate, Stainless Steel (Other Grades), Tygon, Tyvek |
C Fair | Acetal (Delrin), Aluminum (Wet Ozone), Cast Iron, Galvanized Steel, Hypalon, Hytrel, Monel, Neoprene, Polyamide (PA), Polypropylene |
D Poor or Very Poor | Buna-N (Nitrile), Magnesium, Natural Rubber (Very Poor), Nylon (Very Poor), Steel (Carbon, Mild) (Very Poor), Zinc and Zinc Alloy |
EPDM, an oxidation process produces a reduction in molecule size, which generated hydroxyl and carbonyl group [
A disinfection system has been developed using a discharge-based ozone generator. The antibacterial effect was investigated using biological indicators of Gram-stain-positive spore-forming bacteria, Geobacillus stearothermophilus, Bacillus atrophaeus, and Bacillus subtilis; Gram-stain positive bacteria, Staphylococcus aureus, Gram-stain negative bacteria, Pseudomonas aeruginosa and Escherichia coli, the fungi, Aspergillus niger and the yeast, Candida albicans. Disinfection by ozone mixture showed satisfactory results for standard levels of cleanliness, excluding spore-forming bacteria. The sterilization of spore-forming bacteria varied up to a relatively long processing time up to 120 minutes, depending on conditions. In this case, the disinfection system does not meet the standard for the Sterility Assurance Level 6 × Log10 reduction of microorganisms in a limited time period. Efforts are being made to increase the efficiency of the ozone generator to realize processing in a shorter period. The disinfection efficiency is satisfactory for the carrier and adherent microorganisms in sterile packages. Convex and irregularly shaped carrier in the form of the hydrophilic vinyl polysiloxane impression material Exafine and granular acrylic resin can be disinfected. The ozone disinfection system satisfies the requirement for short exposure time and control of the sterility level in medical applications, meeting the need for a more rapid, less damaging, and sterile method.
We acknowledge Mr. Keisuke Ohnishi, Dr. Masao Tsuji and Professor Dr. Masatoshi Ohnishi for collaboration in this experiment. The present work was supported by the Grant-in-Aid for the Development of Innovative Technology (GADIT) 2001-2003, by the Ministry of Education, Science and Culture, Japan.