Plasma Masaaki Nagatsu,* Ying Zhao, Iuliana Motrescu, Ryota Mizutani, Yuya Fujioka, Akihisa Ogino 1. Introduction Conventionally, steamautoclaves are usedto sterilize heat- resistant materials andethyleneoxidesterilizers areusedto sterilize heat-sensitive materials. However, these conven- tional sterilization and disinfection methods suffer from various problems. Inparticular, ethylene oxide sterilization causes environmental problems due to its toxicity. It has thus been desired to develop a new sterilization technique that is capable of sterilizingmedical instruments safelyand rapidly. Full Paper M. Nagatsu, Y. Zhao, I. Motrescu, A. Ogino Graduate School of Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu 432-8561, Japan Fax: 81 53 478 1081; E-mail: tmnagat@ipc.shizuoka.ac.jp M. Nagatsu, R. Mizutani, Y. Fujioka, A. Ogino Graduate School of Engineering, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu 432-8561, Japan I. Motrescu Department of Sciences, The Ion Ionescu De La Brad University of Agricultural Scienecs and Veterinary Medicine, Aleea M. Sadoveanu, Iasi 700490, Romania We demonstrate a novel sterilization technique that sterilizes medical instruments stored in medical containers by generating a microwave-excited volume-wave plasma inside medical containers using a planar microwave launcher. We conrmed that a plasma was generated inside the medical container by the microwaves trans- mitted through the heat-resistant plastic lid of the container. A Langmuir probe was used to study the characteristics of the microwave-excited volume-wave plasma generated inside the container. The inacti- vation characteristics of Geobacillus stearothermophi- lus spores set inside the medical container were also investigated. 2.3 10 6 spores were inactivated after irradiation for 40 min or longer by a plasma generated ina simulated air mixture. This inactivation time could be reduced to 30 min by adding water vapor to the air- simulated plasma. Plasma Process. Polym. 2012, 9, 000000 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com DOI: 10.1002/ppap.201100111 1 Early View Publication; these are NOT the final page numbers, use DOI for citation !! R Various plasma sterilization techniques have been devel- oped that employ low-pressure glow discharge, [1] atmo- spheric-pressure glow discharge, [24] downstream plasma generated by microwave excitation, [5] moving atmospheric microwave plasma [6] and surface-wave plasma. [711] Plasma sterilization methods have several advantages over conventional methods. For example, plasmasterilizationcan be performed at relatively low temperatures and relatively rapidly. However, plasma sterilization techniques are generally useful for sterilizing the surfaces of medical instruments. We recently presented the results of inactiva- tion measurements of biological indicators (BI) sealed by a Tyveksheet usingalow-pressuremicrowave-excitedplasma and showed that 10 6 Geobacillus stearothermophilus spores were inactivated after irradiation for 60min or longer without any thermal damage of Tyvek sheet. [11,12] In the present study, we describe a novel sterilization technique in which a plasma is generated inside a medical container containing medical instruments by microwaves introduced through a plastic lid using a planar microwave launcher. Its inactivation properties were investigated using the spore-forming bacteria, G. stearothermophilus, which was put inside the medical container together with the medical instruments. 2. Experimental Section Todemonstratesterilizationof medical instrumentsinsideamedical container, we designed and fabricated the prototype microwave plasma device shown in Figure 1. We used a planar microwave launcher togenerateamicrowaveplasmainsidethecontainer; it has been described in detail in previous papers. [13,14] In the present microwave launcher, a quartz disk (diameter: 118mm; thickness: 11mm) was attached to a thin stainless-steel plate by screws (see Figure 2). We used a metal medical container (length: 27cm; width: 27cm; height: 15cm) that had a plastic lid (Muranaka Medical Instruments Co.). The lid was made of heat-resistant plastic and could withstand temperatures up to about 1308C. The body of the medical container was made of aluminum alloy. The planar microwavelauncher was attachedtothelidof themedical container by inserting a silicone rubber sheet as a microwave-transparent buffer. The discharge gas was introduced into the medical container through a perforated Tyvek 1 lter tted belowthe plastic lid (black square in Figure 2b). In the present experiment, we used Ar and a nitrogen/oxygengas mixture, whichwas used tosimulate air as the discharge gases. Amicrowave-excited plasma was generated inside the medical container by introducing microwaves. To measure the electron density and temperature, a Langmuir probe witha Cuwire (length: 4mm; diameter: 0.9mm) made witha semi-rigid cable was inserted in the container through a hermeti- cally sealed SubMiniature Type A (SMA) connector attached to the container wall. We used this probe to measure the plasma parameters inside the container, which was lled with Ar gas. Sterilization experiments were performed using BIs. The non- pathogenic spore-forming bacteria, G. stearothermophilus (ATCC# 12980, Raven Biological, US) is commonly used as a sterility indicator; it contains spore populations in the range of 1.9 2.310 6 . In the present study, its spores were pasted on a small rectangular stainless-steel (SUS) plate, which was placed in a Tyvek 1 /polypouch. For colony forming unit (CFU) count, BIs that had not been exposed to plasma were used as a control. Spore survivors fromboththeplasma-exposedandthecontrol BI samples were recovered by plunging the carriers into 1.5ml of brainheart infusionsolutioninatest tube. Test tubes containingtheBI carriers were vortexed for 1 min at room temperature. 0.1ml of the spore suspension from the test tube was inoculated onto nutrient agar media with triple replication. The survivors were counted as the number of CFU per BI carrier after incubation at 55 8C for 24 h. Survival curves were obtained by plotting the CFU counts results. Another simple way to evaluate the inactivationof spores is to use tryptic soybroth(TSB) as a culture solutionandbromocresol purple as a pHindicator. If spores survive, the color of the culture solution changes from purple to yellow after incubation for 24h. 3. Results and Discussion 3.1. Discharge Characteristics of Microwave Plasma Generated in the Container To conrm the plasma generation below the plastic lid attached by a planar microwave launcher, we carried out Figure 1. Photograph and schematic diagram of prototype micro- wave plasma device. 2 Plasma Process. Polym. 2012, 9, 000000 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ppap.201100111 R Early View Publication; these are NOT the final page numbers, use DOI for citation !! M. Nagatsu, Y. Zhao, I. Motrescu, R. Mizutani, Y. Fujioka, A. Ogino the experiments shown in Figure 3. We compared the plasma discharges with and without the plastic lid of the container belowthe planar microwave launcher, as shown inFigure 3a. Photographs of plasma discharges with Ar and air as working at gas pressure of 6.7 Pa and gas owrate of 70 sccmgas are shown in Figure 3b and c, respectively. The incident microwave power was about 300W and reected one was roughly 2030 W. The electron density in the case of Ar plasma was measured by the Langmuir probe located at 5.5cmbelowthe bottomsurface of microwave launcher. In the case without the lid, the electron density was about 2.5 10 11 cm 3 , which was higher than the cutoff density, 7.4 10 10 cm 3 . It is expectedthat the electrondensitynear the microwave launcher will be higher than the critical density of surface-wave, 3.6 10 11 cm 3 . This condition is generally satised in a typical surface-wave plasma generation. On the other hand, in the case with the lid, the density was 5610 10 cm 3 by a factor of 45 lower than the case without the lid, that is, lower than the cutoff density. Such plasma is characteristic of the volume-wave plasma. [15] When we installed the medical container below the microwave launcher witharubber buffer sheet inbetween, microwaves propagated through the plastic lid into the container where they generated a plasma. The plasma discharge inside the medical container could be easily conrmed by using a medical container with a metal mesh sidewall. Figure 4 shows photographs taken before and after turning on the Ar plasma discharge in the medical con- tainer. It also contains a schematic drawing of the container with the mesh sidewall. The plasma was generated using a microwave power of 400W at an Ar gas pressure of 27 Pa and a gas ow rate of 200 sccm. We performed Langmuir probe mea- surements to determine whether a sur- face-wave or volume-wave plasma was generated inside the container. The posi- tion of the probe tip was varied from horizontal to vertical by bending the cable. Figure 5 depicts the probe mea- surement geometry. We dened the center of the container as r 0 and the axial (vertical) position z was measured relative to the bottom of the container, which was dened as z 0. The diagonal direction is dened as the projection of the r axis onto the horizontal plane, as illustrated in Figure 5. As mentioned above, surface-wave excitation by 2.45 GHz microwaves Figure 3. (a) Schematics of experimental geometries and photographs of plasma discharges generated by the planar microwave launcher attached with (right) and without (left) a plastic lid of container in the cases of (b) Ar and (c) air, respectively. Figure 2. Photographs of (a) planar microwave launcher and (b) medical container. Plasma Process. Polym. 2012, 9, 000000 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plasma-polymers.org 3 Early View Publication; these are NOT the final page numbers, use DOI for citation !! R Sterilization Method for Medical Container requires a plasma density higher than the critical density (3.6 10 11 cm 3 ), whereas volume-wave plasmas are generated at plasma densities below the cutoff density (7.4 10 10 cm 3 ). [15] Ar plasmas were generated at various microwave powers in the range 150400 W at a pressure of 40 Pa and a gas ow rate of 100 sccm. Figure 6 shows the dependence of the electron density and electron temperature on the total incident microwave power when the probe was located at r 0 and z 6 cm. The electron temperature is almost constant at about 1.5eV at different microwave powers, whereas the electron density increases approximatelylinearlywiththeincident microwavepower and it appears to be less than the cutoff density. These results suggest that the plasma generated inside the container is probably a volume-wave plasma. Figure 7 shows the electron density distributions along the r-axis at z 6and8cm, whichweremeasuredbyscanningtheprobe in the radial direction. The total incident microwave power was 300W, the Ar gas pressure was 40 Pa, and the gas ow rate was 100sccm. At z 8cm, where the probe is near the lid, theelectrondensitypeakedat thecenter sinceavolume- wave plasmawas generatedimmediatelybelowthe plastic lid, which was beneath the microwave launcher. In contrast, the plasma extends to the container wall at z 6 cm and thus the electron density prole is broader thanthat at z 8cm. Thenext sectionconsiders theeffect of a plasma generated in simulated air mixture on the inactivation of BIs. 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 E l e c t r o n
D e n s i t y
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c m - 3 ) Distance from the Center r (cm) z=8 cm z=6 cm Figure 7. Electron density distributions along the r axis at z 6cm and z 8cm (total incident power: 300W; Ar gas pressure: 40Pa; gas ow rate: 100sccm). Figure 4. Schematic diagram of medical container with an open- structured, mesh sidewall, and photographs before and after Ar plasma discharge inside the medical container. Figure 5. Geometry of Langmuir probe measurements. 0 0.5 1 1.5 2 2.5 0 2 4 6 8 10 150 200 250 300 350 400 E l e c t r o n
T e m p e r a t u r e
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c m - 3 ) Total Incident Power (W) Figure 6. Dependence of electron density and electron tempera- ture at a probe position of r 0 and z 6cm on the total incident microwave power. 4 Plasma Process. Polym. 2012, 9, 000000 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ppap.201100111 R Early View Publication; these are NOT the final page numbers, use DOI for citation !! M. Nagatsu, Y. Zhao, I. Motrescu, R. Mizutani, Y. Fujioka, A. Ogino 3.2. Inactivation of BIs Inside the Container by a Volume-Wave Plasma In an experiment to investigate the inactivation of BIs set inside the container, we employed a simulated air mixture of nitrogen(160 sccm) andoxygen(40 sccm) at apressure of about 90 Pa instead of Ar gas. Amicrowave-excited plasma was generated using time-modulated microwaves with an on-time of 30 s and an off-time of 60 s to prevent thermal damage to the plastic lid of the container; the total microwave power was about 300W. We rst investigated the lethal properties of the microwave-excited volume- wave plasma on BIs by mounting a SUS mesh basket inside the container (see Figure 8). Total plasma irradiation times of 10, 20, 30 and 40 min were used. To investigate the inactivation properties at different positions, we set the BIs at three different positions: in the left corner (BI-1), in the center (BI-2), and in the right corner (BI-3) (see Figure 8). These BIs are located at about z 6 cm. We used heat- sensitive labels (Thermo Label 5E-75, 5E-100 and 5E-125, Nichiyu) to monitor the temperature near the sample position and the back of the plastic lid. Table 1 shows the results obtained for plasma inactivation of G. stearothermophilus using a TSB culture solution with bromocresol purple. After plasma treatment, the samples were incubated at 5560 8C for more than 1 d, which is standard for G. stearothermophilus. The spore mortality was monitored by daily checking the color of the TSB solution during incubation. Figure 9 shows the survival curves obtained for G. stearothermophilus at the center and near the edge of the container. They show that BIs were inactivated after 40 min of plasma treatment. The heat- sensitive labels show that the temperature at the back of the lid in the center of the container was 75 8C<T <80 8C and that near the BI-2 samples was 95 8C<T <1008C after irradiation for 40 min. According to the previous results, [11,12,16] we consider that the main mechanism of bacterial inactivation is the synergetic effect of the etching of bacteria due to oxygen radicals and VUV/UV emission by O atoms, N atoms, NO molecules, andN 2 molecules excitedinthe air plasma. From the morphology analysis using the scanning electron microscopy, it was found that the spores were signicantly eroded by the excited O atoms, which leads to the fatal inactivation of spores. In a previous paper, [12] we reported the effect of addition of water vapor to the simulated air mixture of nitrogenand oxygengases onthesterilitycharacteristics of BIs. Wefound that the addition of a small amount of water vapor caused G. stearothermophilus spores to become inactivated faster than when a dry air gas plasma was used. To demonstrate the effect of the addition of water vapor on the sterilization of the interior of the medical container, we investigatedthe effect of adding water vapor to the simulated air gas mixture. Table 2 and Figure 10, respectively, show the results for TSB culture solution tests and colony counting methods. Both results indicate that 10 6 BI spores were inactivatedapproximately10 minfaster whenwater vapor Table 1. Results for inactivation of G. stearothermophilus in a TSB culture solution by a volume-wave plasma generated in a simu- lated-air mixture. Treatment time [min] BI-1 (left corner) BI-2 (center) BI-3 (right corner) 10 20 30 40 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 10 20 30 40 50 G.stearothermophilus N 2 /O 2 160/40sccm %, %, %, C o l o n y
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( C F U ) Plasma Treatment Time [min] Bl-2 Bl-1 Bl-3 G. stearothermophilus N 2 /O 2 160/40 sccm Figure 9. Survival curves for G. stearothermophilus at center and near the edge after irradiation by a plasma in a simulated air mixture. Figure 8. Photograph of inside of container containing SUS mesh basket indicating the three locations of BIs. Plasma Process. Polym. 2012, 9, 000000 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plasma-polymers.org 5 Early View Publication; these are NOT the final page numbers, use DOI for citation !! R Sterilization Method for Medical Container was added at a partial pressure of 6.8 Pa relative to whenno vapor was added(seeTable1andFigure9). This reductionin the inactivation time may be due to OH radicals produced from the added water vapor acting as strong oxidizing radicals in the inactivation process. Finally, tosimulate arealistic situation, we measuredthe inactivation properties of BIs by placing medical instru- ments, such as forceps, surgical knifes, and tweezers, in the stainless-steel mesh basket (see Figure 11). Although these metallic instruments were inside the container, a plasma could still be easily generated using the same discharge conditions as previously. Figure 12 shows survival curves for BIs at three different positions and roughly z 8 cm. They are similar to those obtained when an empty metal mesh basket was set inside the container. The temperature after treatment for 40 min was 80 8C<T <85 8C at the back of the lid and 75 8C<T <80 8C near the BIs located in the center. These temperatures are slightly lower than those obtained when no medical instruments were present. This may be because the high thermal conductivities of the metallic medical instruments cause them to act heat sinks during plasma treatment. Here, to discuss the heat effect on the inactivation of BIs, we carried out a simple experiment in the atmosphere, where the BIs were put on the temperature-controlled hot plate, where the temperature was kept at 70 and 100 8C withinaripple of 1 8C. Figure 13shows the survival curves of G. stearothermophilus treatedat 70and100 8C. Evenafter 40 min at 1008C, roughly 10 6 spores were still surviving. Table 2. Results for inactivation of G. stearothermophilus in a TSB culture solution by a volume-wave plasma generated in a simu- lated-air mixture to which water vapor had been added. Treatment time [min] BI-1 (left corner) BI-2 (center) BI-3 (right corner) 10 20 30 40 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 10 20 30 40 50 G.stearothermophilus N 2 /O 2 160/40sccm water 75000ppm %, %, %, C o l o n y
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U n i t s ( C F U ) Bl-1 Bl-3 Bl-2 wi th water vapor Plasma Treatment Time (min) G. stearothermophilus N 2 /O 2 160/40 sccm with water vapor Figure 10. Survival curves for G. stearothermophilus at center and near the edge after irradiation by a plasma in a simulated air mixture containing water vapor. Figure 11. Photograph of inside of container containing SUS mesh basket lled with medical instruments indicating the three locations of BIs. Figure 12. Survival curves for G. stearothermophilus at center and near the edge after irradiation by a plasma in a simulated air mixture containing water vapor when the basket contains medical instruments. 6 Plasma Process. Polym. 2012, 9, 000000 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ppap.201100111 R Early View Publication; these are NOT the final page numbers, use DOI for citation !! M. Nagatsu, Y. Zhao, I. Motrescu, R. Mizutani, Y. Fujioka, A. Ogino This indicates that the heat effect on spore inactivation is negligibly small in the proposed sterilization method. 4. Conclusion A novel technique for generating a stable microwave- excited plasma inside a medical container is proposed. Using this technique, medical instruments in a medical container can be readily sterilized without opening the container, just like sterilization using an autoclave. The present experiments conrmed that a plasma could be generated inside a medical container by transmitting microwaves through the heat-resistant plastic lid of the container. The characteristics of microwave-excited volume-wave plasma generated in the container were studied using a Langmuir probe. G. stearothermophilus witha populationof 2.310 6 ina metal meshbasket inthe medical container became inactivatedafter plasma irradia- tion for 40 min or longer by using a plasma in an air- simulated mixture with a gas ow of 200sccm when the total microwave power was 300 W and the pressure was 90 Pa. This was true even when medical instruments were placedinthebasket insidethecontainer. Whenwater vapor was added, the BIs were inactivated about 10 min quicker than when dry simulated air was used. The present experimental results demonstrate rapid (<30 min) sterilizationof the interior of amedical container by a microwave-excited volume-wave plasma at a rela- tively low temperature (<1008C). Acknowledgements: This work was partly supported by a grant- in-aid for Scientic Research from the Japan Society for the Promotion of Science (JSPS). Received: June 5, 2011; Revised: October 15, 2011; Accepted: October 24, 2011; DOI: 10.1002/ppap.201100111 Keywords: low-pressure discharges; microwave discharges; spores; sterilization [1] I. A. Soloshenko, V. V. Tsiolko, V. A. Khomich, A. I. Shchedrin, A. V. Ryabtsev, V. Yu. Bazhenov, I. L. Mikhno, Plasma Phys. Rep. 2000, 26, 792. [2] T. C. Montie, K. Kelly-Wintenberg, J. R. Roth, IEEE Trans. Plasma Sci. 2000, 28, 41. [3] M. Laroussi, I. Alexeff, W. L. Kang, IEEE Trans. Plasma Sci. 2000, 28, 184. [4] V. Y. Bazhenov, A. I. Kuzmichev, V. I. Kryzhanovsky, I. L. Mikhno, A. V. Ryabtsev, I. A. Soloshenko, V. A. Khomich, V. V. Tsiolko, A. I. Shchedrin, Proc. 15th Int. Symp. Plasma Chem. Orleans, France, Vol. II, 2001, 3005. [5] M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, M. Tabriziani, L. H. Yahia, Int. J. Pharm. 2001, 226, 1. [6] J. Ehlbeck, A. Ohl, M. Maas, U. Krohmann, T. Neumann, Surf. Coat. Technol. 2003, 174175, 493. [7] S. Lerouge, M. R. Wertheimer, R. Marchand, M. Tabriziani, L. H. Yahia, J. Biomed. Mater. Res. 2000, 51, 128. [8] M. Nagatsu, F. Terashita, Y. Koide, Jpn. J. Appl. Phys. 2003, 42, L856. [9] M. Nagatsu, F. Terashita, H. Nonaka, L. Xu, T. Nagata, Y. Koide, Appl. Phys. Lett. 2005, 86, 211502. [10] J. Pollak, M. Moisan, D. Keroack, M. K. Boudam, J. Phys. D: Appl. Phys. 2008, 41, 135212. [11] M. K. Singh, A. Ogino, M. Nagatsu, New J. Phys. 2009, 11, 115027. [12] M. K. Singh, A. Ogino, M. Nagatsu, L. Xu, J. Plasma Fus. Res. Ser. 2009, 8, 560. [13] M. Nagatsu, K. Naito, A. Ogino, K. Ninomiya, S. Nanko, Appl. Phys. Lett. 2005, 87, 161501. [14] M. Nagatsu, K. Naito, A. Ogino, S. Nanko, Plasma Sources Sci. Technol. 2006, 15, 37. [15] A. Ogino, K. Naito, F. Terashita, S. Nanko, M. Nagatsu, Jpn. J. Appl. Phys. 2005, 44, L352. [16] Y. Zhao, A. Ogino, M. Nagatsu, Appl. Phys. Lett. 2011, 98, 191501. 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 10 20 30 40 50 C o l o n y
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U n i t s Treatment time (min) 70 degC 100 degC Figure 13. Survival curves for G. stearothermophilus heated at 70 and 1008C. Plasma Process. Polym. 2012, 9, 000000 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plasma-polymers.org 7 Early View Publication; these are NOT the final page numbers, use DOI for citation !! R Sterilization Method for Medical Container