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Sterilization of Escherichia coli and MRSA using microwave-induced argon plasma at atmospheric pressure
Surface & Coatings Technology 193 (2005) 35 – 38 www.elsevier.com/locate/surfcoat
Sterilization of Escherichia coli and MRSA using microwave-induced argon plasma at atmospheric pressure Kwon-Yong Leea, Bong Joo Parkb, Dong Hee Leec, In-Seop Leed, Soon O. Hyune, Kie-Hyung Chungf, Jong-Chul Parkc,g,* a Bioengineering Research Center, Sejong University, 98 Gunja-dong, Gwangjin-gu, Seoul 143-747, Korea The United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan c Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea d Atomic-scale Surface Science Research Center, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea e New Material R and D Center, Korea Institute of Industrial Technology, 35-3 Hongchun-ri, Ibjang-myun, Chunan 330-825, Korea f Physico-Technology Laboratory, Korea Accelerator and Plasma Research Association, Cheorwon-gun, Gangwon-do, Korea g Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, Korea b
Available online 27 August 2004
Abstract The use of microwave plasma for sterilization is a relatively new method. The advantages of this method include relatively low temperature, timesaving and nontoxicity compared to the known techniques, such as dry heat, steam autoclave and ethylene oxide (EtO) gas. The aim of this study was to investigate the sterilization effects on Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) using self-designed, low-cost and reliable, 2.45 GHz, wave guide-based applicator to generate microwave plasma at atmospheric pressure. The results of this study confirmed that the sterilization effect of microwave-induced argon plasma at atmospheric pressure was caused by free radicals and UV light generated during the plasma treatment and the etching process. The microwave plasma system used in this study required much less exposure time than the previous study on bacterial strains of E. coli and MRSA, because of the high plasma density, the large number of free radicals, and the strong UV intensity. D 2004 Elsevier B.V. All rights reserved. Keywords: Sterilization; Escherichia coli; Methicillin-resistant Staphylococcus aureus; Microwave-induced argon plasma; Atmospheric pressure
1. Introduction Escherichia coli remains an important cause of diarrheal disease worldwide [1]. Methicillin-resistant Staphylococcus aureus (MRSA) is a germ that, like many others, can cause an infection, is resistant to most antibiotics, and is increasingly common in hospitals [2,3]. Infection by E. coli as well as MRSA bacteria results in significant public health problem worldwide. Therefore, microbial sterilization is important in the biological and medical fields. Sterilization is based on either a physical or a chemical process that destroys or eliminates microorganisms, or both [4,5]. Traditional methods for sterilization include autoclav-
* Corresponding author. Tel.: +82 2 361 5407; fax: +82 2 363 9923. E-mail address: [email protected] (J.-C. Park). 0257-8972/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2004.07.034
ing, ovens, chemical such as ethylene oxide (EtO), and radiation (gamma rays), which are dependable and well understood. However, all of these methods have their advantages as well as disadvantages. For these reasons, a more rapid and less damaging method of sterilizing various materials is needed [6]. A new sterilization method in the field of protection and conservation of materials from microorganisms is plasma treatment. Sterilization by plasma is an alternative to conventional sterilization methods. Moreover, the advantages of microwave plasma sterilization are the possibility of sterilization at a relatively low temperature, preserving the integrity of polymer-based materials [4], and it is safe as opposed to EtO [5,7]. Moreover, it is not only capable of killing bacteria and viruses, but also capable of removing the dead bacteria and viruses (pyrogens) from the surface of the objects being sterilized [4,8].
36
K.-Y. Lee et al. / Surface & Coatings Technology 193 (2005) 35–38
Fig. 1. A schematic diagram of self-designed, 1 kW, 2.45 GHz microwaveinduced argon plasma system.
This paper describes on the sterilization effects of E. coli and MRSA using microwave-induced argon plasma at atmospheric pressure.
spores were inoculated on slide glasses coated with poly-llysine in Petri dishes, and allowed to dry at room temperature for 30 min. Prior to the sterilization test, the slide glasses inoculated with the bacteria were removed from the Petri dishes and placed in front of a nozzle for the plasma treatment. Both the strains were exposed to the plasma for 1, 2, 3, 4, and 5 s. After plasma treatment, the slide glasses were transferred into a screw cap tube containing 2 ml of 0.9% saline, and shaken for 60 s. The strains in the saline were spread over a standard methods agar plate medium. The number of colonies was counted after 3 days of incubation at 37 8C [9,10]. In addition to colony counts, scanning electron microscopy (SEM) was performed to obtain information regarding the morphologies of the bacteria. The plasma treated and untreated slide glasses were coated with an ultrathin layer of gold/Pt by ion sputter (E1010, HITACHI, Tokyo, Japan), and observed by an electron microscope (S-800, HITACHI).
2. Experimental details 3. Results 2.1. Microwave-induced argon plasma system setup For sterilization test, a microwave-induced argon plasma system to generate plasma at atmospheric pressure was used in this study as in the previous study [9,10]. Fig. 1 shows a schematic diagram of a 2.45 GHz, wave guide-based, microwave-induced argon plasma system. This system consists of a 1 kW magnetron power supply commonly used in a microwave oven, an applicator including a tuning section, which is required to reduce the reflected power, and the nozzle section made of quartz. The plasma generated at the end of a nozzle was formed by an interaction between the high electrical field, which is generated by the microwave power, the wave guide aperture and the gas nozzle. Argon was used as a working gas for this plasma system, which was chosen for its inertness, and the gas flow rate is approximately 100 l per min (lpm) at 8 kgf/cm2.
The sterilization effects of microwave-induced argon plasma at atmospheric pressure on two bacterial strains were shown in Fig. 2. It was revealed that a much shorter plasma treatment time was required than time for bacterial sterilization in our previous study [9,10]. Both bacteria were completely sterilized in less than 1 s regardless of the strain. As shown in the SEM images of E. coli (Fig. 3), the plasma-untreated normal spores (Fig. 3a) had an ellipsoidal shape, while the plasma-exposed E. coli spores for 1, 2, 3, 4, and 5 s (Fig. 3b–f ) were rapidly damaged with holes in the cell walls, a significant reduction in size, and a transformed and amorphous structure (Fig. 3e,f ). Fig. 4 showed the SEM images of MRSA. The plasmauntreated MRSA cells (Fig. 4a) had a globular shape, while the plasma-treated spores (Fig. 4b–f ) were smaller than the untreated controls, and their cell membranes were
2.2. Microorganisms E. coli ATCC 8739 was obtained from the American Type Culture Collection (Rockville, MD, USA) and MRSA was isolated from clinical patients in Yonsei Medical Center at Seoul, Korea. The two bacterial strains were maintained on standard methods agar (Becton, Dickinson and Company, Sparks, MD, USA) slants at 5–6 8C. The cultures were grown on the same medium and kept at 37 8C for 3 days in the dark. 2.3. Sterilization test using microwave-induced argon plasma at atmospheric pressure For the sterilization test, the two bacterial strains were suspended in 0.9% saline. The suspensions of the bacterial
Fig. 2. Sterilization effects of microwave-induced argon plasma at atmospheric pressure against E. coli and MRSA.
K.-Y. Lee et al. / Surface & Coatings Technology 193 (2005) 35–38
37
Fig. 3. SEM photographs of E. coli: untreated control (a) and spores exposed to the microwave-induced argon plasma at atmospheric pressure for 1 (b), 2 (c), 3 (d), 4 (e) and 5 (f ) s.
damaged and ruptured (Fig. 4b–d). Therefore, the cellular contents were released into the surrounding surface. After 4 s of exposure, the MRSA cells were reduced into smaller structures and microscopic debris, as shown in Fig. 4e and f.
4. Discussion Generally, inactivating bacteria from materials is important, but the removal of killed bacteria or their debris from the materials is also important [4]. Therefore, it has been
Fig. 4. SEM photographs of MRSA: untreated control (a) and spores exposed to the microwave-induced argon plasma at atmospheric pressure for 1 (b), 2 (c), 3 (d), 4 (e) and 5 (f ) s.
38
K.-Y. Lee et al. / Surface & Coatings Technology 193 (2005) 35–38
suggested that various plasmas, including microwave plasma, can be used for sterilizing microorganisms [4–7]. In this study, the sterilization effects of microwaveinduced argon plasma at atmospheric pressure on the E. coli and MRSA, disease-causing bacteria [1–3], were evaluated. Two bacteria used were perfectly sterilized in less than 1 s regardless of the strain, as shown in Fig. 2. These results assumed that reactive species and UV light generated by the plasma could diffuse through an otherwise chemically and physically robust membrane more rapidly in bacteria, and directly react with the biomaterials inside the cell. Therefore, both bacteria require significantly less time than previously described other bacteria to be sterilized by the plasma. These results demonstrate the possibility of using microwave-induced argon plasma at atmospheric pressure to sterilize biological and medical materials contaminated with microorganisms. In general, there are three basic mechanisms: DNA destruction by UV irradiation, the erosion of the microorganism through intrinsic photodesorption, and etching (eventually enhanced by UV radiation) in the plasma [5,9– 13]. These processes were confirmed in this study using microwave-induced argon plasma at atmospheric pressure. In this study, the intensity of UV light generated by the plasma ranged from 65 mW/cm2 (minimum) to 94 mW/cm2 (maximum) at a wavelength of 254 nm as in the previous study [9,10], which killed the E. coli and MRSA. Fig. 3 and 4 show SEM images of the E. coli and MRSA spores that were ruptured and damaged by the microwave plasma. The figures show that there was a relatively high level of UV emission in the microwave-induced argon plasma, and that the UV radiation generated was involved in sterilizing the microorganisms. Furthermore, a relatively high level of UV light generated by the plasma enhanced the etching process [9,10]. Another sterilization process of microwave plasma, and one similar to plasma etching, is the erosion of the microorganisms through etching to form volatile compounds, as a result of slow combustion using oxygen atoms or radicals emanating from the plasma. This can be seen in Figs. 3e,f and 4d–f. These SEM images show a significant reduction in size, and exhibit transformed and amorphous morphologies. Therefore, the spores of the E. coli and MRSA were reduced to smaller structures and microscopic debris after plasma exposure. This result demonstrated that a strong etching process of the plasma caused the sterilizing effect on the E. coli and MRSA, when the microorganisms were exposed to this microwave plasma at atmospheric pressure.
5. Conclusion This study confirmed that the sterilization effects of microwave-induced argon plasma at atmospheric pressure on E. coli and MRSA caused by the generation of free radicals, and UV light, as well as the etching process. The microwave plasma system required much less exposure time
than what has been reported from most published systems [4–7,11–23], because of the high plasma density, the large number of free radicals and the strong intensity of generated UV light. These results suggest that this sterilization method is easy to use, requires significantly less exposure time than other methods, such as traditional methods. In addition, it is nontoxic. And, the microwave-induced argon plasma at atmospheric pressure is a powerful sterilization tool for biological and medical materials contaminated with microorganisms.
Acknowledgment This work was supported by Grant No. 02-PJ3-PG1031402-0018 from the Ministry of Health and Welfare of Korea.
References [1] S.C. Clarke, R.D. Haigh, P.P. Freestone, P.H. Williams, Clin. Microbiol. Rev. 16 (2003) 365. [2] D. Metry, R. Katta, Dermatol. Clin. 21 (2003) 269. [3] K. Hiramatsu, L. Cui, M. Kuroda, T. Ito, Trends Microbiol. 9 (2001) 486. [4] T.T. Chau, C.K. Kwan, B. Gregory, M. Francisco, Biomaterials 17 (1996) 1273. [5] N. Philip, B. Saoudi, M.C. Crevier, M. Moisan, J. Barbeau, J. Pelletier, IEEE Trans. Plasma Sci. 30 (2002) 1429. [6] C.M. Thomas, K.W. Kimberly, J.R. Roth, IEEE Trans. Plasma Sci. 28 (2000) 41. [7] A.V. Khomich, I.A. Soloshenko, V.V. Tsiolko, I.L. Mikhno, Proceedings of the 12th International Conference on Gas Discharges and Their Applications, vol. 2, Greifswald, Germany, 1997, p. 740. [8] B.A. Campbell, United States patent 5,184,046 (1993). [9] B.J. Park, D.H. Lee, J.-C. Park, I.-S. Lee, K.-Y. Lee, S.O. Hyun, M.-S. Chun, K.-H. Chung, Phys. Plasmas 10 (2003) 4539. [10] J.-C. Park, B.J. Park, D.-W. Han, D.H. Lee, I.-S. Lee, S.O. Hyun, M.-S. Chun, K.-H. Chung, M. Aihara, K. Takatori, J. Microbiol. Biotechnol. 14 (2004) 188. [11] M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, M. Tabrizian, L.H. Yahia, Int. J. Pharm. 226 (2001) 1. [12] M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, Vide. Sci., Tech. Appl. 299 (2001) 15. [13] M. Laroussi, IEEE Trans. Plasma Sci. 30 (2002) 1409. [14] N.K. Podder, E.D. Mezonlin, J.A. Johnson, IEEE Trans. Plasma Sci. 29 (2001) 965. [15] M. Moisan, Z. Zakrzewski, J. Phys., D. Appl. Phys. 24 (1991) 1025. [16] M. Laroussi, IEEE Trans. Plasma Sci. 24 (1996) 1188. [17] M.L. Elizabeth, Fundamentals of the Fungi, 3rd ed., Prentice-Hall, New Jersey, 1990. [18] M.T. Madigan, J.M. Martinko, J. Parker, Brock Biology of Microorganisms, 8th ed., Prentice-Hall, New Jersey, 1997. [19] D.H. Jeng, K.A. Kaczmarek, A.G. Woodworth, G. Balasky, Microbiology 53 (1987) 2133. [20] G.R. Vela, J.F. Wu, Appl. Environ. Microbiol. 39 (1979) 550. [21] B.A. Welt, C.H. Tong, J.L. Rossen, D.B. Lund, Appl. Environ. Microbiol. 60 (1994) 482. [22] C.M. Thomas, K.W. Kimberly, J.R. Roth, IEEE Trans. Plasma Sci. 28 (2000) 41. [23] D. Purevdorj, N. Igura, M. Shimoda, O. Ariyada, I. Hayakawa, Acta Biotechnol. 21 (2001) 333.
Sterilization of Escherichia coli and MRSA using microwave-induced argon plasma at atmospheric pressure Kwon-Yong Leea, Bong Joo Parkb, Dong Hee Leec, In-Seop Leed, Soon O. Hyune, Kie-Hyung Chungf, Jong-Chul Parkc,g,* a Bioengineering Research Center, Sejong University, 98 Gunja-dong, Gwangjin-gu, Seoul 143-747, Korea The United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan c Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea d Atomic-scale Surface Science Research Center, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea e New Material R and D Center, Korea Institute of Industrial Technology, 35-3 Hongchun-ri, Ibjang-myun, Chunan 330-825, Korea f Physico-Technology Laboratory, Korea Accelerator and Plasma Research Association, Cheorwon-gun, Gangwon-do, Korea g Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, Korea b
Available online 27 August 2004
Abstract The use of microwave plasma for sterilization is a relatively new method. The advantages of this method include relatively low temperature, timesaving and nontoxicity compared to the known techniques, such as dry heat, steam autoclave and ethylene oxide (EtO) gas. The aim of this study was to investigate the sterilization effects on Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) using self-designed, low-cost and reliable, 2.45 GHz, wave guide-based applicator to generate microwave plasma at atmospheric pressure. The results of this study confirmed that the sterilization effect of microwave-induced argon plasma at atmospheric pressure was caused by free radicals and UV light generated during the plasma treatment and the etching process. The microwave plasma system used in this study required much less exposure time than the previous study on bacterial strains of E. coli and MRSA, because of the high plasma density, the large number of free radicals, and the strong UV intensity. D 2004 Elsevier B.V. All rights reserved. Keywords: Sterilization; Escherichia coli; Methicillin-resistant Staphylococcus aureus; Microwave-induced argon plasma; Atmospheric pressure
1. Introduction Escherichia coli remains an important cause of diarrheal disease worldwide [1]. Methicillin-resistant Staphylococcus aureus (MRSA) is a germ that, like many others, can cause an infection, is resistant to most antibiotics, and is increasingly common in hospitals [2,3]. Infection by E. coli as well as MRSA bacteria results in significant public health problem worldwide. Therefore, microbial sterilization is important in the biological and medical fields. Sterilization is based on either a physical or a chemical process that destroys or eliminates microorganisms, or both [4,5]. Traditional methods for sterilization include autoclav-
* Corresponding author. Tel.: +82 2 361 5407; fax: +82 2 363 9923. E-mail address: [email protected] (J.-C. Park). 0257-8972/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2004.07.034
ing, ovens, chemical such as ethylene oxide (EtO), and radiation (gamma rays), which are dependable and well understood. However, all of these methods have their advantages as well as disadvantages. For these reasons, a more rapid and less damaging method of sterilizing various materials is needed [6]. A new sterilization method in the field of protection and conservation of materials from microorganisms is plasma treatment. Sterilization by plasma is an alternative to conventional sterilization methods. Moreover, the advantages of microwave plasma sterilization are the possibility of sterilization at a relatively low temperature, preserving the integrity of polymer-based materials [4], and it is safe as opposed to EtO [5,7]. Moreover, it is not only capable of killing bacteria and viruses, but also capable of removing the dead bacteria and viruses (pyrogens) from the surface of the objects being sterilized [4,8].
36
K.-Y. Lee et al. / Surface & Coatings Technology 193 (2005) 35–38
Fig. 1. A schematic diagram of self-designed, 1 kW, 2.45 GHz microwaveinduced argon plasma system.
This paper describes on the sterilization effects of E. coli and MRSA using microwave-induced argon plasma at atmospheric pressure.
spores were inoculated on slide glasses coated with poly-llysine in Petri dishes, and allowed to dry at room temperature for 30 min. Prior to the sterilization test, the slide glasses inoculated with the bacteria were removed from the Petri dishes and placed in front of a nozzle for the plasma treatment. Both the strains were exposed to the plasma for 1, 2, 3, 4, and 5 s. After plasma treatment, the slide glasses were transferred into a screw cap tube containing 2 ml of 0.9% saline, and shaken for 60 s. The strains in the saline were spread over a standard methods agar plate medium. The number of colonies was counted after 3 days of incubation at 37 8C [9,10]. In addition to colony counts, scanning electron microscopy (SEM) was performed to obtain information regarding the morphologies of the bacteria. The plasma treated and untreated slide glasses were coated with an ultrathin layer of gold/Pt by ion sputter (E1010, HITACHI, Tokyo, Japan), and observed by an electron microscope (S-800, HITACHI).
2. Experimental details 3. Results 2.1. Microwave-induced argon plasma system setup For sterilization test, a microwave-induced argon plasma system to generate plasma at atmospheric pressure was used in this study as in the previous study [9,10]. Fig. 1 shows a schematic diagram of a 2.45 GHz, wave guide-based, microwave-induced argon plasma system. This system consists of a 1 kW magnetron power supply commonly used in a microwave oven, an applicator including a tuning section, which is required to reduce the reflected power, and the nozzle section made of quartz. The plasma generated at the end of a nozzle was formed by an interaction between the high electrical field, which is generated by the microwave power, the wave guide aperture and the gas nozzle. Argon was used as a working gas for this plasma system, which was chosen for its inertness, and the gas flow rate is approximately 100 l per min (lpm) at 8 kgf/cm2.
The sterilization effects of microwave-induced argon plasma at atmospheric pressure on two bacterial strains were shown in Fig. 2. It was revealed that a much shorter plasma treatment time was required than time for bacterial sterilization in our previous study [9,10]. Both bacteria were completely sterilized in less than 1 s regardless of the strain. As shown in the SEM images of E. coli (Fig. 3), the plasma-untreated normal spores (Fig. 3a) had an ellipsoidal shape, while the plasma-exposed E. coli spores for 1, 2, 3, 4, and 5 s (Fig. 3b–f ) were rapidly damaged with holes in the cell walls, a significant reduction in size, and a transformed and amorphous structure (Fig. 3e,f ). Fig. 4 showed the SEM images of MRSA. The plasmauntreated MRSA cells (Fig. 4a) had a globular shape, while the plasma-treated spores (Fig. 4b–f ) were smaller than the untreated controls, and their cell membranes were
2.2. Microorganisms E. coli ATCC 8739 was obtained from the American Type Culture Collection (Rockville, MD, USA) and MRSA was isolated from clinical patients in Yonsei Medical Center at Seoul, Korea. The two bacterial strains were maintained on standard methods agar (Becton, Dickinson and Company, Sparks, MD, USA) slants at 5–6 8C. The cultures were grown on the same medium and kept at 37 8C for 3 days in the dark. 2.3. Sterilization test using microwave-induced argon plasma at atmospheric pressure For the sterilization test, the two bacterial strains were suspended in 0.9% saline. The suspensions of the bacterial
Fig. 2. Sterilization effects of microwave-induced argon plasma at atmospheric pressure against E. coli and MRSA.
K.-Y. Lee et al. / Surface & Coatings Technology 193 (2005) 35–38
37
Fig. 3. SEM photographs of E. coli: untreated control (a) and spores exposed to the microwave-induced argon plasma at atmospheric pressure for 1 (b), 2 (c), 3 (d), 4 (e) and 5 (f ) s.
damaged and ruptured (Fig. 4b–d). Therefore, the cellular contents were released into the surrounding surface. After 4 s of exposure, the MRSA cells were reduced into smaller structures and microscopic debris, as shown in Fig. 4e and f.
4. Discussion Generally, inactivating bacteria from materials is important, but the removal of killed bacteria or their debris from the materials is also important [4]. Therefore, it has been
Fig. 4. SEM photographs of MRSA: untreated control (a) and spores exposed to the microwave-induced argon plasma at atmospheric pressure for 1 (b), 2 (c), 3 (d), 4 (e) and 5 (f ) s.
38
K.-Y. Lee et al. / Surface & Coatings Technology 193 (2005) 35–38
suggested that various plasmas, including microwave plasma, can be used for sterilizing microorganisms [4–7]. In this study, the sterilization effects of microwaveinduced argon plasma at atmospheric pressure on the E. coli and MRSA, disease-causing bacteria [1–3], were evaluated. Two bacteria used were perfectly sterilized in less than 1 s regardless of the strain, as shown in Fig. 2. These results assumed that reactive species and UV light generated by the plasma could diffuse through an otherwise chemically and physically robust membrane more rapidly in bacteria, and directly react with the biomaterials inside the cell. Therefore, both bacteria require significantly less time than previously described other bacteria to be sterilized by the plasma. These results demonstrate the possibility of using microwave-induced argon plasma at atmospheric pressure to sterilize biological and medical materials contaminated with microorganisms. In general, there are three basic mechanisms: DNA destruction by UV irradiation, the erosion of the microorganism through intrinsic photodesorption, and etching (eventually enhanced by UV radiation) in the plasma [5,9– 13]. These processes were confirmed in this study using microwave-induced argon plasma at atmospheric pressure. In this study, the intensity of UV light generated by the plasma ranged from 65 mW/cm2 (minimum) to 94 mW/cm2 (maximum) at a wavelength of 254 nm as in the previous study [9,10], which killed the E. coli and MRSA. Fig. 3 and 4 show SEM images of the E. coli and MRSA spores that were ruptured and damaged by the microwave plasma. The figures show that there was a relatively high level of UV emission in the microwave-induced argon plasma, and that the UV radiation generated was involved in sterilizing the microorganisms. Furthermore, a relatively high level of UV light generated by the plasma enhanced the etching process [9,10]. Another sterilization process of microwave plasma, and one similar to plasma etching, is the erosion of the microorganisms through etching to form volatile compounds, as a result of slow combustion using oxygen atoms or radicals emanating from the plasma. This can be seen in Figs. 3e,f and 4d–f. These SEM images show a significant reduction in size, and exhibit transformed and amorphous morphologies. Therefore, the spores of the E. coli and MRSA were reduced to smaller structures and microscopic debris after plasma exposure. This result demonstrated that a strong etching process of the plasma caused the sterilizing effect on the E. coli and MRSA, when the microorganisms were exposed to this microwave plasma at atmospheric pressure.
5. Conclusion This study confirmed that the sterilization effects of microwave-induced argon plasma at atmospheric pressure on E. coli and MRSA caused by the generation of free radicals, and UV light, as well as the etching process. The microwave plasma system required much less exposure time
than what has been reported from most published systems [4–7,11–23], because of the high plasma density, the large number of free radicals and the strong intensity of generated UV light. These results suggest that this sterilization method is easy to use, requires significantly less exposure time than other methods, such as traditional methods. In addition, it is nontoxic. And, the microwave-induced argon plasma at atmospheric pressure is a powerful sterilization tool for biological and medical materials contaminated with microorganisms.
Acknowledgment This work was supported by Grant No. 02-PJ3-PG1031402-0018 from the Ministry of Health and Welfare of Korea.
References [1] S.C. Clarke, R.D. Haigh, P.P. Freestone, P.H. Williams, Clin. Microbiol. Rev. 16 (2003) 365. [2] D. Metry, R. Katta, Dermatol. Clin. 21 (2003) 269. [3] K. Hiramatsu, L. Cui, M. Kuroda, T. Ito, Trends Microbiol. 9 (2001) 486. [4] T.T. Chau, C.K. Kwan, B. Gregory, M. Francisco, Biomaterials 17 (1996) 1273. [5] N. Philip, B. Saoudi, M.C. Crevier, M. Moisan, J. Barbeau, J. Pelletier, IEEE Trans. Plasma Sci. 30 (2002) 1429. [6] C.M. Thomas, K.W. Kimberly, J.R. Roth, IEEE Trans. Plasma Sci. 28 (2000) 41. [7] A.V. Khomich, I.A. Soloshenko, V.V. Tsiolko, I.L. Mikhno, Proceedings of the 12th International Conference on Gas Discharges and Their Applications, vol. 2, Greifswald, Germany, 1997, p. 740. [8] B.A. Campbell, United States patent 5,184,046 (1993). [9] B.J. Park, D.H. Lee, J.-C. Park, I.-S. Lee, K.-Y. Lee, S.O. Hyun, M.-S. Chun, K.-H. Chung, Phys. Plasmas 10 (2003) 4539. [10] J.-C. Park, B.J. Park, D.-W. Han, D.H. Lee, I.-S. Lee, S.O. Hyun, M.-S. Chun, K.-H. Chung, M. Aihara, K. Takatori, J. Microbiol. Biotechnol. 14 (2004) 188. [11] M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, M. Tabrizian, L.H. Yahia, Int. J. Pharm. 226 (2001) 1. [12] M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, Vide. Sci., Tech. Appl. 299 (2001) 15. [13] M. Laroussi, IEEE Trans. Plasma Sci. 30 (2002) 1409. [14] N.K. Podder, E.D. Mezonlin, J.A. Johnson, IEEE Trans. Plasma Sci. 29 (2001) 965. [15] M. Moisan, Z. Zakrzewski, J. Phys., D. Appl. Phys. 24 (1991) 1025. [16] M. Laroussi, IEEE Trans. Plasma Sci. 24 (1996) 1188. [17] M.L. Elizabeth, Fundamentals of the Fungi, 3rd ed., Prentice-Hall, New Jersey, 1990. [18] M.T. Madigan, J.M. Martinko, J. Parker, Brock Biology of Microorganisms, 8th ed., Prentice-Hall, New Jersey, 1997. [19] D.H. Jeng, K.A. Kaczmarek, A.G. Woodworth, G. Balasky, Microbiology 53 (1987) 2133. [20] G.R. Vela, J.F. Wu, Appl. Environ. Microbiol. 39 (1979) 550. [21] B.A. Welt, C.H. Tong, J.L. Rossen, D.B. Lund, Appl. Environ. Microbiol. 60 (1994) 482. [22] C.M. Thomas, K.W. Kimberly, J.R. Roth, IEEE Trans. Plasma Sci. 28 (2000) 41. [23] D. Purevdorj, N. Igura, M. Shimoda, O. Ariyada, I. Hayakawa, Acta Biotechnol. 21 (2001) 333.