- Email: [email protected]
Plasma sterilization using glow discharge at atmospheric pressure
Surface & Coatings Technology 193 (2005) 29 – 34 www.elsevier.com/locate/surfcoat
Plasma sterilization using glow discharge at atmospheric pressure Tetsuya Akitsua,*, Hiroshi Ohkawaa, Masao Tsujib, Hideo Kimurab, Masuhiro Kogomac a
Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Takeda 4-3-11, Kofu, Yamanashi, Japan b Yamanashi Prefecture Industrial Technology Center, Ohtsu-chou, Kofu, Yamanashi, Japan c Faculty of Science and Technology, Sophia University, Kioi-chou Chiyoda-ku, Tokyo, Japan Available online 9 September 2004
Abstract Recent development of atmospheric pressure glow discharge was compared with the performance of an apparatus used in the first APG experiment, in terms of sterilization of newly classified biological indicator: Bacillus atrophaeus, former Bacillus subtilis var. niger and Geobacillus stearothermophilus. Stabilization was attained by controlling the experimental conditions, at low frequency: 100 kHz and Radio Frequency: 13.56 MHz, water vapor/He dilution. Large volume of meta-stable atomic helium is responsible for the result that aids generation of hydroxyl radicals. D 2004 Elsevier B.V. All rights reserved. Keywords: Atmospheric pressure glow (APG); Plasma sterilization; Bacillus atrophaeus; Geobacillus stearothermophilus
1. Introduction Low temperature plasma sterilization is attracting attentions as safe and short cycle cleaning technique as an alternative method for the chemical sterilization, using ethylene-oxide gas(EOG). Sterilization is inactivation of microorganisms, at factory of medical-care industry and hospital, as a part of prevention of infection and sterility assurance system. Widely accepted conventional schemes can be classified into two categories, high temperature process: pressurized steam treatment, autoclave, and dryheat treatment and low temperature sterilization process: gas sterilization and radiation sterilization. Steam sterilization is suitable for sterilization of metallic and glass-made object. Heat treatment is not suitable for materials that have low resistance to heat. Distortions of plastic parts and deterioration of surgical knives are experienced. Gas sterilization is a treatment using gaseous compound, ethylene oxide. This process allows low temperature disinfection of materials for medical treatment. Currently, this process is carrying the part of central pillar in low
* Corresponding author. E-mail address: [email protected] (T. Akitsu). 0257-8972/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2004.07.042
temperature sterilization process. Nevertheless, residual gaseous agent influences patients and medical operators toxically, and strong concern to human carcinogenicity is expressed since the early 1990s [1,2]. Thus, environmental emission is strongly restricted by PRTR law, Pollutant Release and Transfer Resistor law, 2000, in Japan. Sterilization process using hydrogen-peroxide plasma is a scheme developed in the 1980s, performed by a batch process in vacuum chambers, using hydrogen-peroxide flush followed by back diffusion RF plasma. This scheme was approved by former Ministry of Health and Welfare in Japan in1994, and is coming into wide use as an alternative approach for the ethylene-oxide gas sterilization. In a rigorously scientific definition, major mechanism for sterilization is chemical sterilization by hydrogen peroxide vapor. The present work aims to describe our plasma sterilization system using pulse modulated radio frequency, 27.12 MHz, under the atmospheric pressure. The idea of atmospheric pressure glow was first developed by S. Okazaki and her researchers group. First report was presented in 1987 in The International Symposium on Plasma Chemistry (ISPC-8) Tokyo conference. The atmospheric pressure glow (APG) plasma was first described by Kanazawa et al in 1987, 1988, 1989, Okazaki and Kogoma in 1989 [3–7]. The APG plasma method is applicable to majority part of plasma treatments at
30
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
atmospheric pressures because of its operational low cost compared with the chamber-type, low-pressure plasma. With an attitude to find out new applications for this old knowledge, we compared the sterilizations characteristics of the original apparatus used in the early stage of the APG research. The original apparatus is conserved in good operational condition in Sophia University. Study on the antibacterial effect was carried out using RF glow discharge, at 100 kHz and 13.56 MHz; using two type of spore-forming bacteria: Bacillus atrophaeus and Geobacillus stearothermophilus. Although acceptable RF power is quite limited, for the original apparatus, still we can find out that higher frequency was capable to exhibited shorter cleaning cycle in the terms of the antibacterial effect. New system can deliver pulse modulated RF power at 27.12 MHz, up to 1 kW, to larger volume, realizing shorter sterilization time without thermal deterioration. In these experiments, helium gas, working gas, has principal effect for the homogeneously excited plasma at high pressures, because of its low breakdown voltage. Helium atoms have high-energy meta-stable states. The energy transfer excitation is responsible for the expansion of the micro-discharges on the insulating plate. The excitation of mixed water molecules to generate free-radicals, such as hydroxyl radicals He þ electronYHe4ð23 S; 21 SÞ þ electron He4 þ H2 OYHe þ HO þ H and atomic oxygen. He þ electronYHe4ð23 S; 21 SÞ þ electron
Fig. 1. Experimental setup for the APG plasma sterilization. (a) Electrodes, glass vessel and storage for distilled water for the vapor flow. (b) The circuit diagram and a schematic presentation for the structure of the electrodes (Original APG apparatus).
He4 þ O2 YHe þ O4 þ O These oxygen radicals react with the cell-wall of microorganism and destroy its double helix structure of DNA.
2. Experimental apparatus In the original version, the electrical discharge was generated between plane metallic electrodes covered with optical flat glass, 50 mm in diameter. The gap between the electrodes was 5 mm. The entire vacuum system is constructed using glass tube and vessels. Fig. 1 shows the experimental setup. Plasma was excited at two frequency ranges: audio-frequency range 100 kHz, 80 W, and radio frequency range 13.56 MHz, 150–200 W. These two operational conditions were selected as typical examples for low damage, moderate treatment for soft materials such as cellulose sheet and short treatment for heat-resisting materials such as metallic tool; at 13.56 MHz. These conditions were selected as one of best parameters for each process considering the limited heat-resistance of glass vessel and electrodes. Our intention is not the empirical discovery of power scaling law.
Temperature of the glass (insulator) covering the electrode was measured with Infra-Red Thermometer through a BaF2 optical window. Saturated water vapor was supplied as source for reactive oxygen radicals. New application for an experimental comparison, a pulse-modulated RF APG, at 27.12 MHz, was constructed (Fig. 2). Anti-bacterial effect was validated using biological indicators: spore-forming bacteria: B. atrophaeus ATCC9372 and G. stearothermophilus ATCC7953, and selected species of opportunistic pathogen: Escherichia coli ATCC8739, Salmonella enteritidis, Staphylococcus aureus ATCC6538, Candida albicans ATCC10231. The discharge volume between two dielectric barrier discharge electrodes has a dimension of 150 mm in length, 50 mm in width, and 3 mm gap The whole installation consists of linear actuator for programmable transportation of samples of microorganisms and a RF power generator, 670 W, at industrial frequency: 27.12 MHz. Electrodes are cooled by circulation of chilled water circulating inside the aluminum blocks. Metallic parts of upper and lower electrodes were covered with transparent fused quartz plate of size, 230 mm in length115 mm in width3 mm in thickness. Dielectric
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
31
Fig. 2. Schematic drawing for developed pulse-modulated RF-APG device, at 27.12 MHz.
barriers play important roles in prevention of arcing and in the so-called non-thermal excitation of the plasma. Volume of the discharge region was surrounded by heat-resistant glass and removable shutter made of synthetic rubber. Working gas mixture of helium and oxygen was supplied from array of apertures of size, 1 mm in diameter. The flow rate of working gas was controlled with mass-flow controllers, in a range of 1 ml to 5 l/min. Small amount of oxygen, 0.06%, was mixed as source for reactive oxygen radicals.
3. Experimental result Table 1 shows the experimental result for the first experiment, at 100 kHz, 80 W, and the reflected power range 16–19 W with manual matching circuit. The biological indicator was prepared with a cellulose carrier, a soft contaminating material of 630 mm in dimension containing spores of Bacillus subtilis or Bacillus stearothermophilus. The gas flow rates was He 443 ml/min in 1–6, and additional 2000 ml/min was supplied in 7–9. Water vapor was carried by one of gas flow controller system of 443 ml/
Table 1 Antibacterial effect of APG at 100 kHz
1 2 3 4 5 6 7 8 9
Exposure tyvec/ direct
Flow rate (ml/min)
Exposure (min)
Biological indicator
Result
Direct Direct Direct Tyvek Tyvek Tyvek Direct Tyvek Direct
443 443 443 443 443 443 2000+443 2000+443 2000+443
30 16 5 30 16 5 30 30 30
B. subtilis B. subtilis B. subtilis B. subtilis B. subtilis B. subtilis B. subtilis B. subtilis B. stearothermophilus
+ + + + -
min at 3.2 mol%. Additional helium gas stabilized the discharge, and resulted in observable improvement of homogeneity. In cases 1–8, the biological indicator was spores of B. atrophaeus (former B. subtilis var. niger) ATCC 9372, 2.0106 CFU. In the case no. 9, Geobcillus stearothermophilus ATCC 7953, 1.3106 CFU. In the incubation and sterility judgments, we followed protocols in the Japanese Pharmacopeias. Incubation was performed in Soy been Casein Digest (SCD) culture medium, 100 ml. The incubation temperature was 30–35 8C for B. atrophaeus, and 55–57 8C for G. stearothermophilus. The negative mark indicates sterilization when no visible turbidity of the SCD medium was observed. In nos. 1, 4, 7, 8 and 9, the biological indicator was exposed either direct or indirect packaged form for 30 min. The results in nos. 2, 3, 5 and 6, indicate experimental results in insufficient shorter exposure. Case no. 9 showed sterilization of G. stearothermophilus. The experimental result indicated that the sterilization can be achieved in 30 min in low frequency APG, in low power density conditions either in directly exposed or in-directly in a Tyvek case of BI. In, APG excited with RF power, at 13.56 MHz, a biological indicator was prepared with spores of B. stearothermophilus applied on stainless-steel carrier covered by Tyvek package. The aim of this experiment is shorter sterilization of metallic surgical devices such as surgical knives and scissors, etc. The incidental RF power was 150 and 200 W. The flow rate was 2663 ml/min (typical value). Water vapor was carried by one of gas flow controller system of 663 ml/min at 3.2 mol%. Additional 2000 ml/min was mixed for cooling. The results are shown in Table 2. The experimental result reads that the sterilization can be achieved in 5 min. Discovery of some difficulties should be described. In the sterilization experiment using APG plasma at 100 kHz, a columnar structure of the plasma was observed. Fig. 3 shows a typical example for the structure. The columnar structure indicates the localized enhancement
32
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
Table 2 Antibacterial effect of APG plasma at 13.56 MHz
1 2 3 4 5 6 7
Power (W)
Exposure tyvec/direct
Exposure time (min)
Result
150 150 150 200 200 200 200
Tyvek Tyvek Tyvek Tyvek Tyvek Direct Direct
10 10 5 3 3 3 3
+ -
of the excitation by the plasma. The discharge phenomena left localized enhancement of oxidation of the package, as shown in Fig. 3b. At higher frequencies, at 13.56 MHz, excitation of free radicals in the plasma is more intensive
Fig. 3. The columnar structure developed by ionization instabilities and localized damage; (a) the APG plasma discharge, (b) pin-holes left on the biological indicator and (c) brighter APG plasma at 13.56 MHz excitation.
and the antibacterial effect was strong enough to realize the sterilization in shorter exposure, and no columnar plasma structure was observed. The temperature of the plasma and the electrode surface was measured with IR thermometer. Because the temperature of the center of the driven electrode is invisible due to the IR radiation from the high temperature edge, the temperature of the central part was measured with a thermocouple immediately after the discharge was terminated. The temperature of the upper electrode increased to 108 8C and the lower electrode was 167 8C. The central region of the upper electrode was cooled by a contact of the water pipe and the surface was maintained below 47 8C. At 100 kHz the measurement indicated 59 8C, thus the low
Fig. 4. Temporal evolution of the electrode and wall temperature. Open circles indicates the temperature of the upper electrode, triangles the wall; (a) 100 kHz, (b) 13.56 MHz (original APG apparatus).
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
33
Table 3 Correlation between oxygen ratio and antibacterial effect for B. atrophaeus Oxygen/helium (%)
Flow rate (L/minute)
Scan time per each surface (minutes)
He
O2
2.0
2.5
3.0
3.5
4.0
0 0.06 0.13 0.25 0.50 1.00
1.5 1.5 1.5 1.5 1.5 1.5
0 0.001 0.0019 0.0038 0.0075 0.015
+ + +
+ + + +
+ + + + +
+ +
+ +
4.5
5.0
10.0
-
+ -
+ -
-
*Biological indicator: Attestk1264, 3M (B. atrophaeus). **RF power: 670W, Pulse width and interval: 10 micro second.
frequency APG meets the request of low temperature treatment in the entire discharge region. On the other hand, at 13.56 MHz, the present requirement meets near by the central part. The biological indicator was suspended from the center of the driven electrode (Fig. 4). In the following part, the antibacterial effect of pulsemodulated RF-APG was measured using biological indicators applied on glass slide covered with Tyvek sterile package. The power source is pulse-modulated 27.12 MHz, 670W. Experimental result is shown in Tables 3–5. In the incubation and sterility judgments, we followed protocols in the Japanese Pharmacopeias [8]. After the plasma treatment, cover glass was picked out of Tyvek package and incubated in 100 ml of SCD fluid culture medium. B stearothermophilus was incubated at 5560 8C. C. albicans was incubated at 25 8C, and other microorganisms were incubated at 30–35 8C, for 7 days. Validation of the salinity was judged on the basis of turbidity of the culture medium Culture medium remains clear only if 6 log10 grade sterilization was performed successfully. Commercially available biological indicators of spore forming bacteria Attestk 1262 and AttestTM 1264, 3M were also used for a comparative study. Each capsule contains G. stearothermophilus ATCC7953, 8.6105 CFU, and B. atrophaeus ATCC9372, 4.8106 CFU, respectively. Incubation was carried out for 48 h in vials and validation of salinity was judged by a change of color of the incubation medium by pH indicator. G. stearothermophilus is approved
Table 4 Dependence of disinfection time on oxygen ration for Staphylococcus aureus Oxygen/helium (%)
Flow rate L/minute
Processing time (minutes)
He
O2
1
2
3
4
0 0.06 0.25
1.5 1.5 1.5
0 1 3.8
+ + +
+ + +
+ +
+ -
* +: Propagation of bacteria was observed, -: No propagation was observed. ** Experimental conditions: RF power: 670 W, Frequency: 27.12 MHz, Gap: 3 mm, Pulse width: and intervals: 10 micro seconds, Gas temperature: 90 degrees C. *** Biological indicator: Staphylococcus aureus ATCC 6538 (1.3108 CFU) applied and dried on glass slide.
as a standard indicator for steam sterilization, and B. atrophaeus is an approved indicator for gas sterilization using ethylene oxide. The pulse modulation of RF power, with pulse width and interval, 10 As each, enabled the control of neutral gas temperature during the disinfection process lower than 90 8C. The experimental result shows that the sterilization of spore forming bacteria was possible in somewhat shorter without visible deterioration of Tyvek sheet surrounding the biological indicators, and selected species of opportunistic pathogen can be disinfected within much shorter time. The problem of the gas heating was controlled successfully.
4. Conclusion In the site of medical treatment: Surgery, nursing, and transportation of medical-care products, industrial production of food and kitchen, prevention of infection is principle technology for human life. Current stage of disinfection is realized using combination of multiple method including, dry heat, high-temperature vapor, auto-craving, gas-sterilization using ethylene-oxide, UV and radiation sterilization. Low-grade disinfection using alcohol, aldehyde, sodiumhypo chloride also play important roles. Request for novel sterilization scheme is attracting attentions. Plasma sterilization is attracting attentions as one of novel scheme for sterilization and atomic level cleaning. The anti-bacterial effect was examined using the original APG device using helium diluted vapor plasma. In the present experiment, standard 106 Log sterilization of B. atrophaeus and G. stearothermophilus bacterium was successfully observed. This apparatus can show disinfection capability for other type of bacteria including pathogen, because B. atrophaeus shows higher resistance compared to other bacteria. In the disinfection experiment using biological indicators in sterile packaging, the new application showed reasonable disinfection time: 1 min for E. coli, S. enteritidis and C. albicans, 5 min for S. aureus, and 20 min for sporeforming bacteria: B. atrophaeus and G. stearothermophilus,
34
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
Table 5 Disinfection of non-spore forming microorganisms in Tyvek package Species and density (CFU)
Plasma treatment (minutes)
Heat treatment minutes, 90 degrees C)
1
3
5
10
15
1
3
5
10
15
Escherichia coli ATCC8739 (1.6107) Salmonella enteritidis (3.5107) Staphylococcus aureus ATCC6538 (4.7107) Candida albicans ATCC10231 (5.1106)
+ -
+ -
-
-
-
+ + + +
+ + -
+ + -
+ -
+ -
* Biological indicator: Glass slide carrier. ** RF power: 670 W, Pulse width and interval: 10 Asec. *** He:1.5 L/min, O2:1 mL/min.
at the best condition for oxygen radical production, at oxygen/helium ratio of 0.06%. The plasma treatment can also be applied to the surface treatment such as improvement on surfaces of polymer materials for stronger adhesive strength. Present system attained an advantage of continuous processing over batch processing of plasma treatment. Finally, we developed a laboratory fit for plasma sterilization. As an appraisal standard in the experiment, disinfection time was measured using variety of biological indicators of microorganisms including spore-forming bacteria and selected species of pathogen. Notable remarks of the present work are: 1.
2.
3.
Antibacterial effect of atmospheric plasma was tested using an original apparatus used in the early stage of the APG study, and newly developed high power RF-APG device. The present system uses nontoxic mixture of helium and water vapor or oxygen, operated under the atmospheric pressure. No need for vacuum chambers allowing continuous loading of objects. The relative amount of oxygen radicals, atomic oxygen varied depending on oxygen/helium ratio The maximum was observed when oxygen/helium ratio was 0.06%, helium flow rate 1.5 l/min and oxygen flow rate 1 ml/min) The shortest disinfection time was obtained at the same mixture ratio for a biological indicator, S. aureus. The disinfection time was measured with biological indicators applied on to no woven sheet. Complete sterilization was achieved in 1 min for E. coli, S. enteritidis and C. albicans, 5 min for S. aureus, and 20 min for spore-forming bacteria: B. atrophaeus and G. stearothermophilus.
In order to find out technological leap between the original APG-device and our recent laboratory development of the pulse-modulated, validation test was carried out using the APG apparatus developed in the early stage of the research of plasma under the atmospheric pressure. Pulse-modulated RFAPG technique enables the control of gas temperature leaving the input RF power at relatively high level.
Acknowledgement The present work was supported by the Grant in aid for the development of innovative technology by the Ministry of Education, Science and Culture, Japan.
References [1] ATSDR (Agency for Toxic Substances and Disease Registry) Medical Management Guidelines (MMGs) for Ethylene Oxide ([CH2]2O) GAS 75-21-8 UN#1040, August 22, 2003. [2] Current Intelligence Bulletin 35, dEthylene Oxide (EtO)T DHHS Publication No. 81-130, May 22,1981. [3] S. Kanazawa, M. Kogoma, T. Moriwaki, S. Okazaki, Proceedings of ISPC-8 (1987, Tokyo) p. 146 and p. 1839, also in J. Phys. D: Appl. Phys. 21 (1988) 838. [4] S. Kanazawa, M. Kogoma, S. Okazaki, T. Moriwaki, Nucl. Instrum. Methods Phys. Res., B Beam Interact. Mater. Atoms 37138 (1989) 842. [5] A. Nagata, S. Takehiro, S. Sumi, M. Kogoma, S. Okazaki, Y. Horiike, Proceedings of Japan Symp. Plasma Chemistry, vol. 2, 1989, p. 109. [6] S. Okazaki, M. Kogoma, Proceedings of the 2nd Int. Symp. High Pressure Low Temperature Plasma Chemistry, 1989, p. 13, (Kazimirz, Poland). [7] T. Yokoyama, M. Kogoma, T. Moriwaki, S. Okazaki, J. Phys. D: Appl. Phys. 23 (1990) 1125. [8] The 14th edition of Japanese Pharmacopoeias, 1, Tokyo, Japan, 2001, pp. B-628, in Japanese.
Plasma sterilization using glow discharge at atmospheric pressure Tetsuya Akitsua,*, Hiroshi Ohkawaa, Masao Tsujib, Hideo Kimurab, Masuhiro Kogomac a
Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Takeda 4-3-11, Kofu, Yamanashi, Japan b Yamanashi Prefecture Industrial Technology Center, Ohtsu-chou, Kofu, Yamanashi, Japan c Faculty of Science and Technology, Sophia University, Kioi-chou Chiyoda-ku, Tokyo, Japan Available online 9 September 2004
Abstract Recent development of atmospheric pressure glow discharge was compared with the performance of an apparatus used in the first APG experiment, in terms of sterilization of newly classified biological indicator: Bacillus atrophaeus, former Bacillus subtilis var. niger and Geobacillus stearothermophilus. Stabilization was attained by controlling the experimental conditions, at low frequency: 100 kHz and Radio Frequency: 13.56 MHz, water vapor/He dilution. Large volume of meta-stable atomic helium is responsible for the result that aids generation of hydroxyl radicals. D 2004 Elsevier B.V. All rights reserved. Keywords: Atmospheric pressure glow (APG); Plasma sterilization; Bacillus atrophaeus; Geobacillus stearothermophilus
1. Introduction Low temperature plasma sterilization is attracting attentions as safe and short cycle cleaning technique as an alternative method for the chemical sterilization, using ethylene-oxide gas(EOG). Sterilization is inactivation of microorganisms, at factory of medical-care industry and hospital, as a part of prevention of infection and sterility assurance system. Widely accepted conventional schemes can be classified into two categories, high temperature process: pressurized steam treatment, autoclave, and dryheat treatment and low temperature sterilization process: gas sterilization and radiation sterilization. Steam sterilization is suitable for sterilization of metallic and glass-made object. Heat treatment is not suitable for materials that have low resistance to heat. Distortions of plastic parts and deterioration of surgical knives are experienced. Gas sterilization is a treatment using gaseous compound, ethylene oxide. This process allows low temperature disinfection of materials for medical treatment. Currently, this process is carrying the part of central pillar in low
* Corresponding author. E-mail address: [email protected] (T. Akitsu). 0257-8972/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2004.07.042
temperature sterilization process. Nevertheless, residual gaseous agent influences patients and medical operators toxically, and strong concern to human carcinogenicity is expressed since the early 1990s [1,2]. Thus, environmental emission is strongly restricted by PRTR law, Pollutant Release and Transfer Resistor law, 2000, in Japan. Sterilization process using hydrogen-peroxide plasma is a scheme developed in the 1980s, performed by a batch process in vacuum chambers, using hydrogen-peroxide flush followed by back diffusion RF plasma. This scheme was approved by former Ministry of Health and Welfare in Japan in1994, and is coming into wide use as an alternative approach for the ethylene-oxide gas sterilization. In a rigorously scientific definition, major mechanism for sterilization is chemical sterilization by hydrogen peroxide vapor. The present work aims to describe our plasma sterilization system using pulse modulated radio frequency, 27.12 MHz, under the atmospheric pressure. The idea of atmospheric pressure glow was first developed by S. Okazaki and her researchers group. First report was presented in 1987 in The International Symposium on Plasma Chemistry (ISPC-8) Tokyo conference. The atmospheric pressure glow (APG) plasma was first described by Kanazawa et al in 1987, 1988, 1989, Okazaki and Kogoma in 1989 [3–7]. The APG plasma method is applicable to majority part of plasma treatments at
30
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
atmospheric pressures because of its operational low cost compared with the chamber-type, low-pressure plasma. With an attitude to find out new applications for this old knowledge, we compared the sterilizations characteristics of the original apparatus used in the early stage of the APG research. The original apparatus is conserved in good operational condition in Sophia University. Study on the antibacterial effect was carried out using RF glow discharge, at 100 kHz and 13.56 MHz; using two type of spore-forming bacteria: Bacillus atrophaeus and Geobacillus stearothermophilus. Although acceptable RF power is quite limited, for the original apparatus, still we can find out that higher frequency was capable to exhibited shorter cleaning cycle in the terms of the antibacterial effect. New system can deliver pulse modulated RF power at 27.12 MHz, up to 1 kW, to larger volume, realizing shorter sterilization time without thermal deterioration. In these experiments, helium gas, working gas, has principal effect for the homogeneously excited plasma at high pressures, because of its low breakdown voltage. Helium atoms have high-energy meta-stable states. The energy transfer excitation is responsible for the expansion of the micro-discharges on the insulating plate. The excitation of mixed water molecules to generate free-radicals, such as hydroxyl radicals He þ electronYHe4ð23 S; 21 SÞ þ electron He4 þ H2 OYHe þ HO þ H and atomic oxygen. He þ electronYHe4ð23 S; 21 SÞ þ electron
Fig. 1. Experimental setup for the APG plasma sterilization. (a) Electrodes, glass vessel and storage for distilled water for the vapor flow. (b) The circuit diagram and a schematic presentation for the structure of the electrodes (Original APG apparatus).
He4 þ O2 YHe þ O4 þ O These oxygen radicals react with the cell-wall of microorganism and destroy its double helix structure of DNA.
2. Experimental apparatus In the original version, the electrical discharge was generated between plane metallic electrodes covered with optical flat glass, 50 mm in diameter. The gap between the electrodes was 5 mm. The entire vacuum system is constructed using glass tube and vessels. Fig. 1 shows the experimental setup. Plasma was excited at two frequency ranges: audio-frequency range 100 kHz, 80 W, and radio frequency range 13.56 MHz, 150–200 W. These two operational conditions were selected as typical examples for low damage, moderate treatment for soft materials such as cellulose sheet and short treatment for heat-resisting materials such as metallic tool; at 13.56 MHz. These conditions were selected as one of best parameters for each process considering the limited heat-resistance of glass vessel and electrodes. Our intention is not the empirical discovery of power scaling law.
Temperature of the glass (insulator) covering the electrode was measured with Infra-Red Thermometer through a BaF2 optical window. Saturated water vapor was supplied as source for reactive oxygen radicals. New application for an experimental comparison, a pulse-modulated RF APG, at 27.12 MHz, was constructed (Fig. 2). Anti-bacterial effect was validated using biological indicators: spore-forming bacteria: B. atrophaeus ATCC9372 and G. stearothermophilus ATCC7953, and selected species of opportunistic pathogen: Escherichia coli ATCC8739, Salmonella enteritidis, Staphylococcus aureus ATCC6538, Candida albicans ATCC10231. The discharge volume between two dielectric barrier discharge electrodes has a dimension of 150 mm in length, 50 mm in width, and 3 mm gap The whole installation consists of linear actuator for programmable transportation of samples of microorganisms and a RF power generator, 670 W, at industrial frequency: 27.12 MHz. Electrodes are cooled by circulation of chilled water circulating inside the aluminum blocks. Metallic parts of upper and lower electrodes were covered with transparent fused quartz plate of size, 230 mm in length115 mm in width3 mm in thickness. Dielectric
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
31
Fig. 2. Schematic drawing for developed pulse-modulated RF-APG device, at 27.12 MHz.
barriers play important roles in prevention of arcing and in the so-called non-thermal excitation of the plasma. Volume of the discharge region was surrounded by heat-resistant glass and removable shutter made of synthetic rubber. Working gas mixture of helium and oxygen was supplied from array of apertures of size, 1 mm in diameter. The flow rate of working gas was controlled with mass-flow controllers, in a range of 1 ml to 5 l/min. Small amount of oxygen, 0.06%, was mixed as source for reactive oxygen radicals.
3. Experimental result Table 1 shows the experimental result for the first experiment, at 100 kHz, 80 W, and the reflected power range 16–19 W with manual matching circuit. The biological indicator was prepared with a cellulose carrier, a soft contaminating material of 630 mm in dimension containing spores of Bacillus subtilis or Bacillus stearothermophilus. The gas flow rates was He 443 ml/min in 1–6, and additional 2000 ml/min was supplied in 7–9. Water vapor was carried by one of gas flow controller system of 443 ml/
Table 1 Antibacterial effect of APG at 100 kHz
1 2 3 4 5 6 7 8 9
Exposure tyvec/ direct
Flow rate (ml/min)
Exposure (min)
Biological indicator
Result
Direct Direct Direct Tyvek Tyvek Tyvek Direct Tyvek Direct
443 443 443 443 443 443 2000+443 2000+443 2000+443
30 16 5 30 16 5 30 30 30
B. subtilis B. subtilis B. subtilis B. subtilis B. subtilis B. subtilis B. subtilis B. subtilis B. stearothermophilus
+ + + + -
min at 3.2 mol%. Additional helium gas stabilized the discharge, and resulted in observable improvement of homogeneity. In cases 1–8, the biological indicator was spores of B. atrophaeus (former B. subtilis var. niger) ATCC 9372, 2.0106 CFU. In the case no. 9, Geobcillus stearothermophilus ATCC 7953, 1.3106 CFU. In the incubation and sterility judgments, we followed protocols in the Japanese Pharmacopeias. Incubation was performed in Soy been Casein Digest (SCD) culture medium, 100 ml. The incubation temperature was 30–35 8C for B. atrophaeus, and 55–57 8C for G. stearothermophilus. The negative mark indicates sterilization when no visible turbidity of the SCD medium was observed. In nos. 1, 4, 7, 8 and 9, the biological indicator was exposed either direct or indirect packaged form for 30 min. The results in nos. 2, 3, 5 and 6, indicate experimental results in insufficient shorter exposure. Case no. 9 showed sterilization of G. stearothermophilus. The experimental result indicated that the sterilization can be achieved in 30 min in low frequency APG, in low power density conditions either in directly exposed or in-directly in a Tyvek case of BI. In, APG excited with RF power, at 13.56 MHz, a biological indicator was prepared with spores of B. stearothermophilus applied on stainless-steel carrier covered by Tyvek package. The aim of this experiment is shorter sterilization of metallic surgical devices such as surgical knives and scissors, etc. The incidental RF power was 150 and 200 W. The flow rate was 2663 ml/min (typical value). Water vapor was carried by one of gas flow controller system of 663 ml/min at 3.2 mol%. Additional 2000 ml/min was mixed for cooling. The results are shown in Table 2. The experimental result reads that the sterilization can be achieved in 5 min. Discovery of some difficulties should be described. In the sterilization experiment using APG plasma at 100 kHz, a columnar structure of the plasma was observed. Fig. 3 shows a typical example for the structure. The columnar structure indicates the localized enhancement
32
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
Table 2 Antibacterial effect of APG plasma at 13.56 MHz
1 2 3 4 5 6 7
Power (W)
Exposure tyvec/direct
Exposure time (min)
Result
150 150 150 200 200 200 200
Tyvek Tyvek Tyvek Tyvek Tyvek Direct Direct
10 10 5 3 3 3 3
+ -
of the excitation by the plasma. The discharge phenomena left localized enhancement of oxidation of the package, as shown in Fig. 3b. At higher frequencies, at 13.56 MHz, excitation of free radicals in the plasma is more intensive
Fig. 3. The columnar structure developed by ionization instabilities and localized damage; (a) the APG plasma discharge, (b) pin-holes left on the biological indicator and (c) brighter APG plasma at 13.56 MHz excitation.
and the antibacterial effect was strong enough to realize the sterilization in shorter exposure, and no columnar plasma structure was observed. The temperature of the plasma and the electrode surface was measured with IR thermometer. Because the temperature of the center of the driven electrode is invisible due to the IR radiation from the high temperature edge, the temperature of the central part was measured with a thermocouple immediately after the discharge was terminated. The temperature of the upper electrode increased to 108 8C and the lower electrode was 167 8C. The central region of the upper electrode was cooled by a contact of the water pipe and the surface was maintained below 47 8C. At 100 kHz the measurement indicated 59 8C, thus the low
Fig. 4. Temporal evolution of the electrode and wall temperature. Open circles indicates the temperature of the upper electrode, triangles the wall; (a) 100 kHz, (b) 13.56 MHz (original APG apparatus).
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
33
Table 3 Correlation between oxygen ratio and antibacterial effect for B. atrophaeus Oxygen/helium (%)
Flow rate (L/minute)
Scan time per each surface (minutes)
He
O2
2.0
2.5
3.0
3.5
4.0
0 0.06 0.13 0.25 0.50 1.00
1.5 1.5 1.5 1.5 1.5 1.5
0 0.001 0.0019 0.0038 0.0075 0.015
+ + +
+ + + +
+ + + + +
+ +
+ +
4.5
5.0
10.0
-
+ -
+ -
-
*Biological indicator: Attestk1264, 3M (B. atrophaeus). **RF power: 670W, Pulse width and interval: 10 micro second.
frequency APG meets the request of low temperature treatment in the entire discharge region. On the other hand, at 13.56 MHz, the present requirement meets near by the central part. The biological indicator was suspended from the center of the driven electrode (Fig. 4). In the following part, the antibacterial effect of pulsemodulated RF-APG was measured using biological indicators applied on glass slide covered with Tyvek sterile package. The power source is pulse-modulated 27.12 MHz, 670W. Experimental result is shown in Tables 3–5. In the incubation and sterility judgments, we followed protocols in the Japanese Pharmacopeias [8]. After the plasma treatment, cover glass was picked out of Tyvek package and incubated in 100 ml of SCD fluid culture medium. B stearothermophilus was incubated at 5560 8C. C. albicans was incubated at 25 8C, and other microorganisms were incubated at 30–35 8C, for 7 days. Validation of the salinity was judged on the basis of turbidity of the culture medium Culture medium remains clear only if 6 log10 grade sterilization was performed successfully. Commercially available biological indicators of spore forming bacteria Attestk 1262 and AttestTM 1264, 3M were also used for a comparative study. Each capsule contains G. stearothermophilus ATCC7953, 8.6105 CFU, and B. atrophaeus ATCC9372, 4.8106 CFU, respectively. Incubation was carried out for 48 h in vials and validation of salinity was judged by a change of color of the incubation medium by pH indicator. G. stearothermophilus is approved
Table 4 Dependence of disinfection time on oxygen ration for Staphylococcus aureus Oxygen/helium (%)
Flow rate L/minute
Processing time (minutes)
He
O2
1
2
3
4
0 0.06 0.25
1.5 1.5 1.5
0 1 3.8
+ + +
+ + +
+ +
+ -
* +: Propagation of bacteria was observed, -: No propagation was observed. ** Experimental conditions: RF power: 670 W, Frequency: 27.12 MHz, Gap: 3 mm, Pulse width: and intervals: 10 micro seconds, Gas temperature: 90 degrees C. *** Biological indicator: Staphylococcus aureus ATCC 6538 (1.3108 CFU) applied and dried on glass slide.
as a standard indicator for steam sterilization, and B. atrophaeus is an approved indicator for gas sterilization using ethylene oxide. The pulse modulation of RF power, with pulse width and interval, 10 As each, enabled the control of neutral gas temperature during the disinfection process lower than 90 8C. The experimental result shows that the sterilization of spore forming bacteria was possible in somewhat shorter without visible deterioration of Tyvek sheet surrounding the biological indicators, and selected species of opportunistic pathogen can be disinfected within much shorter time. The problem of the gas heating was controlled successfully.
4. Conclusion In the site of medical treatment: Surgery, nursing, and transportation of medical-care products, industrial production of food and kitchen, prevention of infection is principle technology for human life. Current stage of disinfection is realized using combination of multiple method including, dry heat, high-temperature vapor, auto-craving, gas-sterilization using ethylene-oxide, UV and radiation sterilization. Low-grade disinfection using alcohol, aldehyde, sodiumhypo chloride also play important roles. Request for novel sterilization scheme is attracting attentions. Plasma sterilization is attracting attentions as one of novel scheme for sterilization and atomic level cleaning. The anti-bacterial effect was examined using the original APG device using helium diluted vapor plasma. In the present experiment, standard 106 Log sterilization of B. atrophaeus and G. stearothermophilus bacterium was successfully observed. This apparatus can show disinfection capability for other type of bacteria including pathogen, because B. atrophaeus shows higher resistance compared to other bacteria. In the disinfection experiment using biological indicators in sterile packaging, the new application showed reasonable disinfection time: 1 min for E. coli, S. enteritidis and C. albicans, 5 min for S. aureus, and 20 min for sporeforming bacteria: B. atrophaeus and G. stearothermophilus,
34
T. Akitsu et al. / Surface & Coatings Technology 193 (2005) 29–34
Table 5 Disinfection of non-spore forming microorganisms in Tyvek package Species and density (CFU)
Plasma treatment (minutes)
Heat treatment minutes, 90 degrees C)
1
3
5
10
15
1
3
5
10
15
Escherichia coli ATCC8739 (1.6107) Salmonella enteritidis (3.5107) Staphylococcus aureus ATCC6538 (4.7107) Candida albicans ATCC10231 (5.1106)
+ -
+ -
-
-
-
+ + + +
+ + -
+ + -
+ -
+ -
* Biological indicator: Glass slide carrier. ** RF power: 670 W, Pulse width and interval: 10 Asec. *** He:1.5 L/min, O2:1 mL/min.
at the best condition for oxygen radical production, at oxygen/helium ratio of 0.06%. The plasma treatment can also be applied to the surface treatment such as improvement on surfaces of polymer materials for stronger adhesive strength. Present system attained an advantage of continuous processing over batch processing of plasma treatment. Finally, we developed a laboratory fit for plasma sterilization. As an appraisal standard in the experiment, disinfection time was measured using variety of biological indicators of microorganisms including spore-forming bacteria and selected species of pathogen. Notable remarks of the present work are: 1.
2.
3.
Antibacterial effect of atmospheric plasma was tested using an original apparatus used in the early stage of the APG study, and newly developed high power RF-APG device. The present system uses nontoxic mixture of helium and water vapor or oxygen, operated under the atmospheric pressure. No need for vacuum chambers allowing continuous loading of objects. The relative amount of oxygen radicals, atomic oxygen varied depending on oxygen/helium ratio The maximum was observed when oxygen/helium ratio was 0.06%, helium flow rate 1.5 l/min and oxygen flow rate 1 ml/min) The shortest disinfection time was obtained at the same mixture ratio for a biological indicator, S. aureus. The disinfection time was measured with biological indicators applied on to no woven sheet. Complete sterilization was achieved in 1 min for E. coli, S. enteritidis and C. albicans, 5 min for S. aureus, and 20 min for spore-forming bacteria: B. atrophaeus and G. stearothermophilus.
In order to find out technological leap between the original APG-device and our recent laboratory development of the pulse-modulated, validation test was carried out using the APG apparatus developed in the early stage of the research of plasma under the atmospheric pressure. Pulse-modulated RFAPG technique enables the control of gas temperature leaving the input RF power at relatively high level.
Acknowledgement The present work was supported by the Grant in aid for the development of innovative technology by the Ministry of Education, Science and Culture, Japan.
References [1] ATSDR (Agency for Toxic Substances and Disease Registry) Medical Management Guidelines (MMGs) for Ethylene Oxide ([CH2]2O) GAS 75-21-8 UN#1040, August 22, 2003. [2] Current Intelligence Bulletin 35, dEthylene Oxide (EtO)T DHHS Publication No. 81-130, May 22,1981. [3] S. Kanazawa, M. Kogoma, T. Moriwaki, S. Okazaki, Proceedings of ISPC-8 (1987, Tokyo) p. 146 and p. 1839, also in J. Phys. D: Appl. Phys. 21 (1988) 838. [4] S. Kanazawa, M. Kogoma, S. Okazaki, T. Moriwaki, Nucl. Instrum. Methods Phys. Res., B Beam Interact. Mater. Atoms 37138 (1989) 842. [5] A. Nagata, S. Takehiro, S. Sumi, M. Kogoma, S. Okazaki, Y. Horiike, Proceedings of Japan Symp. Plasma Chemistry, vol. 2, 1989, p. 109. [6] S. Okazaki, M. Kogoma, Proceedings of the 2nd Int. Symp. High Pressure Low Temperature Plasma Chemistry, 1989, p. 13, (Kazimirz, Poland). [7] T. Yokoyama, M. Kogoma, T. Moriwaki, S. Okazaki, J. Phys. D: Appl. Phys. 23 (1990) 1125. [8] The 14th edition of Japanese Pharmacopoeias, 1, Tokyo, Japan, 2001, pp. B-628, in Japanese.