- Email: [email protected]
Sterilization of microorganisms in silk fabrics by microwave-induced argon plasma treatment at atmospheric pressure
Surface & Coatings Technology 202 (2008) 5773–5778
Contents lists available at ScienceDirect
Surface & Coatings Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s u r f c o a t
Sterilization of microorganisms in silk fabrics by microwave-induced argon plasma treatment at atmospheric pressure Dong Jeong Park a,b, Mi Hee Lee a,b, Yeon I Woo a,b, Dong-Wook Han c, Jae Bong Choi d, Jeong Koo Kim e, Soon O. Hyun f, Kie-Hyung Chung f, Jong-Chul Park a,b,⁎ a
Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea Department of Nanomedical Engineering, College of Nanoscience & Nanotechnology, Pusan National University, Busan 609-735, South Korea d Division of Industrial and Systems Engineering, 389 Samsung-dong 2-ga, Sungbuk-gu, Hansung University, Seoul 136-792, South Korea e Department of Biomedical Engineering, College of Biomedical Science and Engineering, Inje University, 607 Obang-dong, Gimhae 621-749, South Korea f Physico-Technology Laboratory, Korea Accelerator and Plasma Research Association, 377-3 Jadeung-ri, Seo-myun, Cheorwon-gun, Gangwon-do 269-843, South Korea b c
A R T I C L E
I N F O
Available online 9 June 2008 Keywords: Silk fabrics Microbial contamination Sterilization Microwave-induced argon plasma Ultimate tensile strength
A B S T R A C T Since many old cultural assets, such as clothes, pictures and books, remain in the state of silk fabrics or papers, it is very difficult as well as important to retain their original form. Microbial contamination induces surface deformations and strength degradation of silk fabrics by invading deeply into the fibers. In this study, the sterilization effects of microwave-induced argon plasma at atmospheric pressure were investigated on the microorganisms in silk fabrics. Also, the influence of the plasma treatment was examined on the physical properties of the fabrics. Argon plasma treatment completely sterilized the silk fabrics inoculated with various strains of either bacteria or fungi. It was also found that the plasma treatment did not affect the ultimate tensile strength and surface morphology of the fabrics because it took advantage of relatively low temperature. As increase in the plasma treatment, however, the lightness of silk fabrics decreased with concomitant increased in the color intensities of green and yellow. These results suggest that microwaveinduced argon plasma can be effectively used as an alternative method for sterilizing and protecting cultural assets out of silk fabrics without any deterioration of the tensile strength and surface morphology. © 2008 Elsevier B.V. All rights reserved.
1. Introduction It is well known that the main cause of damage to fabrics is microbial growth. Such problem is more serious for organic fabrics or fabrics dyed with organic materials than for inorganic compounds [1]. Microorganisms on silk fabrics cause not only surface deformations, but also strength degradation by invading deeply into the fibers. When the microbial infection of silks occurs, it results in the degradation of the fabrics and the color change of them through microbial secretions or spores [2,3]. In addition, such attached microorganisms can deteriorate the physical structure and property of the silk fibers. 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 autoclaving, dry heat, ethylene oxide (EG) gas, gamma ray and UV irradiation, which are reliable and well understood. However, all of these methods have their advan⁎ Corresponding author. Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea. Tel.: +82 2 2228 1917; fax: +82 2 363 9923. E-mail address: [email protected] (J.-C. Park). 0257-8972/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2008.06.039
tages as well as their disadvantages. Heat treatment methods may not be suitable for low-melting point materials such as polymerbased materials [6]. An EO treatment is highly toxic, which can be absorbed in plastics [7]. Radiation may also cause the material to undergo undesirable changes during sterilization [8]. For these reasons, a more rapid and less damaging method of sterilizing various materials is needed. Sterilization by plasma is an alternative method to these conventional sterilization methods. Plasma treatment is a new sterilization method in the field of protection and conservation of materials from microorganisms. Moreover, microwave-induced argon plasma treatment has the advantages, such as the sterilizing potential at a relatively low temperature, the possible preservation of the integrity of polymer-based materials and the biological safety compared to EO gas [5,6,9]. In addition, it is not only capable of killing bacteria and fungi, but also able to remove the dead microbes (e.g., pyrogens) from the surface of the objects to be sterilized [10,11]. The purpose of this study is to investigate whether microwaveinduced argon plasma at atmospheric pressure can sterilize the microorganisms in silk fabrics or not. Also, the influence of the plasma treatment was examined on the physical properties of the fabrics for longer term conservation.
5774
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
2. Experimental details 2.1. Microwave-induced argon plasma system As previously described [2,5], a 2.45 GHz, waveguide-based, microwave-induced argon plasma system was used to generate plasma at atmospheric pressure. In brief, this system consists of a 1 kW magnetron power supply used in a microwave oven, a WR-284 copper waveguide and an applicator including a tuning and a nozzle section. The plasma generated at the end of a nozzle was formed by an interaction between the high electrical fields. Argon was used as a working gas for this plasma system, which was chosen due to its inertness, and the gas flow rate is about 100 l/min at 6 kgf/cm2. 2.2. Microbial strains Bacterial strains, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6358p, were obtained from the American Type Culture Collection (Rockville, MD). They were maintained on standard methods agar (Bacto™, BD Diagnostic Systems, Sparks, MD) at 4 °C. Before few days of inoculation, the strains were transferred onto new agar plates and cultured at 37 °C. Fungal strains used in this study, Aspergillus niger Yonsei Medical Center (YMC) 0100 and Penicillium citrinum YMC 0175, were isolated from indoor dust and air and then kept as stock cultures [14]. These strains were maintained on potato dextrose agar (Difco, Detroit, MI) slants with 30 μl/l chloramphenicol (Wako Pure Chemical Industries, Ltd., Osaka, Japan) to suppress bacterial contamination and the cultures were cultured at 25 °C for 7 days prior to use. 2.3. Sterilization of silk fabrics by plasma treatment Silk fabrics used in this study were kindly supported from Dr. Soon O. Hyon (Psysico-Technolgy Laboratory, Korea Accelerator and Plasma Reserch Association) and prepared as the dimension of 0.9 cm wide × 30 cm long × 0.2 cm thick. For the sterilization test, the microbial strains were inoculated in a 0.9% saline solution. The silk fabrics were inoculated with each microbial suspension and then dried at room temperature for 30 min. The fabrics inoculated with each strain were placed in front of a nozzle from which plasma was blown and exposed to plasma for 1–7 s. After plasma treatment, the fabrics were transferred into a screw-cap tube containing 5 ml saline solution and thoroughly shaken by a vortex for 1 min. The plasmatreated strains were then spread over either a standard methods agar plate for bacteria culture or a potato-dextrose agar plate for fungi culture. The number of recovered colonies was counted as colony forming units (CFU)/ml after either 3 days of incubation at 37 °C for bacteria growth or 7 days of incubation at 25 °C for fungi growth. In addition to colony counts, scanning electron microscopy (SEM) was performed to obtain detailed information regarding the morphological alterations of the strains of P. aeruginosa and A. niger as previously described [2,5]. The plasma-treated and non-treated silk fabrics were sputter-coated with gold/platinum using an ion coater (E1010, Hitachi, Tokyo, Japan) and observed under an electron microscope (S-800, Hitachi) at an accelerating voltage of 20 kV.
(MTS 858, MTS Systems Co., Eden Prairie, MN). A displacement of 10 mm/min was applied to the fabrics along with the longitudinal direction, and all data were collected at a frequency of 20 Hz until break occurred. 2.4.2. SEM observation The surface morphology of silk fabrics treated without or with microwave-induced argon plasma was observed under a scanning electron microscope (Hitachi S-800) as mentioned above. 2.4.3. Evaluation of color change of silk fabrics using L⁎a⁎b⁎ color coordinate system For evaluating the color changes of silk fabrics, the fabrics was prepared as a disk of 0.9 cm in diameter and 0.2 cm in thickness and treated with plasma for 1–5 s. After plasma treatment, the color change was analyzed by the L⁎a⁎b⁎ [L⁎: lightness (or luminance), a⁎: balance between green (−) and red (+), b⁎: balance between blue (−) and yellow (+)] color coordinate system in combination with a personal computer as previously described [13]. Briefly, the L⁎a⁎b⁎ color difference between the non-treated and plasma-treated fabrics was measured at four different points on each fabric with a central cross stripe connected to a computer. All variables were tested in three independent measurements, which were repeated twice (n = 24). Quantitative data were expressed as mean± standard deviation. Statistical comparisons were carried out with repeated ANOVA (SPSS 12.0 for windows). A value of p b 0.05 was considered statistically significant. 3. Results and discussion 3.1. Sterilization effects of plasma treatment on microorganisms in silk fabrics The sterilization effects of microwave-induced argon plasma at atmospheric pressure on bacterial and fungal strains in silk fabrics are shown in Fig. 1. Although S. aureus was the most resistant bacterial strain to the plasma treatment, all the strains were completely sterilized in less than 7 s. The number of recovered colonies after plasma treatment was significantly decreased in a time-dependent manner. In our previous studies, it has already been shown that several bacterial and fungal strains can be fully sterilized and inactivated by plasma treatment within 20 s or less [2,5,6]. Therefore, it is suggested that microwave-induced argon plasma at atmospheric pressure can sterilize silk fabrics contaminated with microorganisms. Recent studies have reported that the plasma-treated textiles or fabrics showed a significant increase in the incorporation of oxygencontaining groups (e.g., C–O, O–CfO and CfO), which allowed the
2.4. Effects of plasma treatment on physical properties of silk fabrics 2.4.1. Mechanical test In order to determine the influence of plasma treatment on the mechanical properties of silk fabrics, the tensile strength at break of the fabrics was measured before and after plasma treatment for 1–5 s. The dimension of the testing fabrics was prepared equally to that of the fabrics used for sterilization test. According to the guidelines of ASTM D5035-06 [12], the ultimate tensile strength of plasma-treated or non-treated fabrics was determined using a material testing system
Fig. 1. Sterilization effects of microwave-induced argon plasma treatment on silk fabrics inoculated with some kinds of bacterial and fungal strains.
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
surface of them more reactive and hydrophilic [14,15]. It would seem that the formation of hydrophilic oxygen-containing functional groups on the textiles might lead to enhanced microbial growth on the fiber surface over a period of time. However, the problem of recontamination of the fabrics would be resolved if they were kept after plasma treatment at the place of which the environmental conditions, including humidity, temperature and air, were controllable. 3.2. Morphological observations of plasma-treated microorganisms in silk fabrics The sterilization effect of plasma treatment was confirmed by SEM micrographs showing the morphological alterations of the strains of P. aeruginosa (Fig. 2) and A. niger (Fig. 3) by plasma treatment for 5 s. The morphology of the non-treated P. aeruginosa spores was a typically round shape with average dimensions of 0.6 μm in diameter (Fig. 2A and B). In contrast, the plasma-treated spores were completely destroyed with severely damaged and ruptured cell membranes
5775
(Fig. 2C). Moreover, the intracellular contents were all released into the surrounding surface after plasma treatment and the original shape and structure of spores disappeared with microscopic debris (Fig. 2D). Fig. 3 shows the SEM images of A. niger before and after plasma treatment. It was revealed that the non-treated normal spores of A. niger had a globular shape (Fig. 3A and B), while the plasma-treated spores for 5 s were clearly damaged with holes in the cell walls (an insert in Fig. 3C), significant reduction in size as well as a transformed and amorphous morphology (Fig. 3D). It is well known that two combined mechanisms are involved in the plasma-mediated sterilization as follows: the destruction of cell wall and DNA by UV irradiation and activated free radicals, and the erosion of microorganisms through etching eventually enhanced by UV radiation [5,16,17]. The UV and activated free radicals generated during plasma treatment weaken the cell wall of the microorganisms by reacting with the hydrocarbon bonds and cause the disruption of unsaturated bonds, particularly the purine and pyrimidine components of the nucleoproteins. As the process continues, the plasma
Fig. 2. SEM micrographs of morphology of P. aeruginosa in silk fabrics before (A and B) and after (C and D) microwave-induced argon plasma treatment for 5 s.
5776
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
Fig. 3. SEM micrographs of morphology of A. niger in silk fabrics before (A and B) and after (C and D) microwave-induced argon plasma treatment for 5 s.
removes the outer cell wall layer of the microorganisms with its burst and destruction. Another sterilization process of microwave plasma is the erosion of the microorganisms through etching to form volatile compounds through slow combustion by oxygen atoms or radicals emanating from the plasma. 3.3. Effects of plasma treatment on tensile strength and surface morphology of silk fabrics To investigate the effects of microwave-induced argon plasma treatment on the mechanical property of silk fabrics, the tensile strength at break was determined according to the guidelines of ASTM. Mechanical analysis revealed that plasma treatment did not adversely affect the tensile strength of silk fabrics (Fig. 4A). The ultimate tensile strength of the fabrics was slightly decreased by plasma treatment, but there was no significant difference between the non-treated and plasma-treated fabrics. Poll et al. also demonstrated that plasma was able to change the textile surface properties without affecting the interior of the fibers [18]. Microwave-induced plasma by
the atmospheric press gas discharge is a source of electrons, ions, excited atoms and molecules, active free radicals and UV radiation. These factors allow the plasma to function as a unique sterilizing agent for the protection and conservation of various materials, including polymers, papers and fabrics [17]. The major advantage of microwaveinduced plasma treatment to papers or fabrics is the potential of cleaning and sterilization without deterioration of their mechanical strength [19]. The surface morphologies of the non-treated and plasma-treated silk fabrics were then observed by SEM (Fig. 4B–E). It was revealed that plasma treatment did not alter the surface morphology of the fabrics. The plasma-treated fabrics were shown to have almost similar morphology to that of the non-treated fabrics. 3.4. Effects of plasma treatment on color change of silk fabrics To examine the effects of microwave-induced argon plasma treatment on the color change of silk fabrics, the lightness (L⁎), balance (a⁎) between green and red, and balance (b⁎) between blue
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
5777
Fig. 4. Effects of microwave-induced argon plasma treatment on tensile strength and surface morphology of silk fabrics. (A) Change of ultimate tensile strength of the fabrics according to the plasma treatment time. SEM micrographs of the fabrics treated without (B and C) or with (D and E) microwave-induced argon plasma for 5 s.
5778
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
plasma treatment (Fig. 5A). It was also found that the color intensities of green and yellow were significantly (p b 0.05) increased after 4 s of plasma treatment (Fig. 5B and C). It has been already reported that the L⁎a⁎b⁎ color coordinate system was very objective and quantitative and could be useful for the evaluation of treatment effects on colored skin lesions [20]. 4. Conclusions In the present study, it was shown that microwave-induced argon plasma treatment could completely sterilize the silk fabrics contaminated with various strains of microorganisms. It was also found that the plasma treatment did not affect the ultimate tensile strength and surface morphology of the fabrics whereas it decreased the lightness of the fabrics with concomitant increases in the color intensities of green and yellow. These results suggest that microwaveinduced argon plasma can be effectively used as an alternative method for sterilizing and protecting cultural assets out of silk fabrics. Acknowledgements This work was supported by Korea Science and Engineering Foundation (KOSEF, Grant No. R01-2007-000-20472-0). References [1] S.R. Goode, K.W. Baughman, Appl. Spectrosc. 38 (1984) 755. [2] 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. [3] N. Philip, B. Saoudi, M-.C. Crevier, M. Moisan, J. Barbeau, J. Pelletier, IEEE Trans. Plasma Sci. 30 (2002) 1429. [4] A.V. Khomich, I.A. Soloshenko, V.V. Tsiolko, I.L. Mikhno, Proc. of the 12th International Conference on Gas discharges & Their Applications 2 (1997) 740. [5] 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. [6] B.J. Park, K. Takatori, M.H. Lee, D.-W. Han, Y.I. Woo, H.J. Son, J.K. Kim, K.-H. Chung, S.O. Hyun, J.-C. Park, Surf. Coat. Technol. 201 (2007) 5738. [7] A.D. Lucas, K. Merritt, V.M. Hitchins, T.O. Woods, S.G. McNamee, D.B. Lyle, S.A. Brown, J. Biomed. Mater. Res. B Appl. Biomater. 66 (2003) 548. [8] Y. Henon, Med. Device Technol. 3 (1992) 30. [9] T.T. Chau, C.K. Kwan, B. Gregory, M. Fransisco, Biomaterials 17 (1996) 1273. [10] K.-Y. Lee, B.J. Park, D.H. Lee, I.-S. Lee, S.O. Hyun, K.-H. Chung, J.-C. Park, Surf. Coat. Technol. 193 (2005) 35. [11] B.J. Park, K. Takatori, Y. Sugita-Konishi, I.-H. Kim, M.H. Lee, D.-W. Han, K.-H. Chung, S.O. Hyun, J.-C. Park, Surf. Coat. Technol. 201 (2007) 5733. [12] Standard Test Method for Breaking Force and Elongation of Textile Fabrics (Strip Method), ASTM D5035-06 American Society for Testing and Materials, Vol. 07.02 (2000). [13] S.C. Kim, D.W. Kim, J.P. Hong, D.K. Rah, Yonsei Med. J. 41 (2000) 333. [14] R. Morent, N. De Geyter, C. Leys, L. Gengembre, E. Payen, Text. Res. J. 77 (2007) 471. [15] Y.Q. Zhu, E.T. Kang, K.G. Neoh, L. Chan, D.M.Y. Lai, A.C.H. Huan, Appl. Surf. Sci. 225 (2004) 144. [16] M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, M. Tabrizian, L.H. Yahia, Int. J. Pharm. 226 (2001) 1. [17] M. Laroussi, IEEE Trans. Plasma Sci. 30 (2002) 1409. [18] H.U. Poll, U. Schladitz, S. Schreiter, Surf. Coat. Technol. 142/144 (2001) 489. [19] U. Vohrer, I. Trick, J. Bernhardt, C. Oehr, H. Brunner, Surf. Coat. Technol. 142/144 (2001) 1069. [20] D.K. Rah, S.C. Kim, K.H. Lee, B.Y. Park, D.W. Kim, Plast. Reconstr. Surg. 108 (2001) 842.
Fig. 5. Effects of microwave-induced argon plasma treatment on color change of silk fabrics (*p b 0.05 vs. the non-treated, analyzed by repeated ANOVA, n = 24).
and yellow of the fabrics were analyzed using the L⁎a⁎b⁎ color coordinates. It was shown that the lightness of silk fabrics was significantly (p b 0.05) decreased in a time-dependent manner by
Contents lists available at ScienceDirect
Surface & Coatings Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s u r f c o a t
Sterilization of microorganisms in silk fabrics by microwave-induced argon plasma treatment at atmospheric pressure Dong Jeong Park a,b, Mi Hee Lee a,b, Yeon I Woo a,b, Dong-Wook Han c, Jae Bong Choi d, Jeong Koo Kim e, Soon O. Hyun f, Kie-Hyung Chung f, Jong-Chul Park a,b,⁎ a
Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea Department of Nanomedical Engineering, College of Nanoscience & Nanotechnology, Pusan National University, Busan 609-735, South Korea d Division of Industrial and Systems Engineering, 389 Samsung-dong 2-ga, Sungbuk-gu, Hansung University, Seoul 136-792, South Korea e Department of Biomedical Engineering, College of Biomedical Science and Engineering, Inje University, 607 Obang-dong, Gimhae 621-749, South Korea f Physico-Technology Laboratory, Korea Accelerator and Plasma Research Association, 377-3 Jadeung-ri, Seo-myun, Cheorwon-gun, Gangwon-do 269-843, South Korea b c
A R T I C L E
I N F O
Available online 9 June 2008 Keywords: Silk fabrics Microbial contamination Sterilization Microwave-induced argon plasma Ultimate tensile strength
A B S T R A C T Since many old cultural assets, such as clothes, pictures and books, remain in the state of silk fabrics or papers, it is very difficult as well as important to retain their original form. Microbial contamination induces surface deformations and strength degradation of silk fabrics by invading deeply into the fibers. In this study, the sterilization effects of microwave-induced argon plasma at atmospheric pressure were investigated on the microorganisms in silk fabrics. Also, the influence of the plasma treatment was examined on the physical properties of the fabrics. Argon plasma treatment completely sterilized the silk fabrics inoculated with various strains of either bacteria or fungi. It was also found that the plasma treatment did not affect the ultimate tensile strength and surface morphology of the fabrics because it took advantage of relatively low temperature. As increase in the plasma treatment, however, the lightness of silk fabrics decreased with concomitant increased in the color intensities of green and yellow. These results suggest that microwaveinduced argon plasma can be effectively used as an alternative method for sterilizing and protecting cultural assets out of silk fabrics without any deterioration of the tensile strength and surface morphology. © 2008 Elsevier B.V. All rights reserved.
1. Introduction It is well known that the main cause of damage to fabrics is microbial growth. Such problem is more serious for organic fabrics or fabrics dyed with organic materials than for inorganic compounds [1]. Microorganisms on silk fabrics cause not only surface deformations, but also strength degradation by invading deeply into the fibers. When the microbial infection of silks occurs, it results in the degradation of the fabrics and the color change of them through microbial secretions or spores [2,3]. In addition, such attached microorganisms can deteriorate the physical structure and property of the silk fibers. 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 autoclaving, dry heat, ethylene oxide (EG) gas, gamma ray and UV irradiation, which are reliable and well understood. However, all of these methods have their advan⁎ Corresponding author. Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea. Tel.: +82 2 2228 1917; fax: +82 2 363 9923. E-mail address: [email protected] (J.-C. Park). 0257-8972/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2008.06.039
tages as well as their disadvantages. Heat treatment methods may not be suitable for low-melting point materials such as polymerbased materials [6]. An EO treatment is highly toxic, which can be absorbed in plastics [7]. Radiation may also cause the material to undergo undesirable changes during sterilization [8]. For these reasons, a more rapid and less damaging method of sterilizing various materials is needed. Sterilization by plasma is an alternative method to these conventional sterilization methods. Plasma treatment is a new sterilization method in the field of protection and conservation of materials from microorganisms. Moreover, microwave-induced argon plasma treatment has the advantages, such as the sterilizing potential at a relatively low temperature, the possible preservation of the integrity of polymer-based materials and the biological safety compared to EO gas [5,6,9]. In addition, it is not only capable of killing bacteria and fungi, but also able to remove the dead microbes (e.g., pyrogens) from the surface of the objects to be sterilized [10,11]. The purpose of this study is to investigate whether microwaveinduced argon plasma at atmospheric pressure can sterilize the microorganisms in silk fabrics or not. Also, the influence of the plasma treatment was examined on the physical properties of the fabrics for longer term conservation.
5774
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
2. Experimental details 2.1. Microwave-induced argon plasma system As previously described [2,5], a 2.45 GHz, waveguide-based, microwave-induced argon plasma system was used to generate plasma at atmospheric pressure. In brief, this system consists of a 1 kW magnetron power supply used in a microwave oven, a WR-284 copper waveguide and an applicator including a tuning and a nozzle section. The plasma generated at the end of a nozzle was formed by an interaction between the high electrical fields. Argon was used as a working gas for this plasma system, which was chosen due to its inertness, and the gas flow rate is about 100 l/min at 6 kgf/cm2. 2.2. Microbial strains Bacterial strains, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6358p, were obtained from the American Type Culture Collection (Rockville, MD). They were maintained on standard methods agar (Bacto™, BD Diagnostic Systems, Sparks, MD) at 4 °C. Before few days of inoculation, the strains were transferred onto new agar plates and cultured at 37 °C. Fungal strains used in this study, Aspergillus niger Yonsei Medical Center (YMC) 0100 and Penicillium citrinum YMC 0175, were isolated from indoor dust and air and then kept as stock cultures [14]. These strains were maintained on potato dextrose agar (Difco, Detroit, MI) slants with 30 μl/l chloramphenicol (Wako Pure Chemical Industries, Ltd., Osaka, Japan) to suppress bacterial contamination and the cultures were cultured at 25 °C for 7 days prior to use. 2.3. Sterilization of silk fabrics by plasma treatment Silk fabrics used in this study were kindly supported from Dr. Soon O. Hyon (Psysico-Technolgy Laboratory, Korea Accelerator and Plasma Reserch Association) and prepared as the dimension of 0.9 cm wide × 30 cm long × 0.2 cm thick. For the sterilization test, the microbial strains were inoculated in a 0.9% saline solution. The silk fabrics were inoculated with each microbial suspension and then dried at room temperature for 30 min. The fabrics inoculated with each strain were placed in front of a nozzle from which plasma was blown and exposed to plasma for 1–7 s. After plasma treatment, the fabrics were transferred into a screw-cap tube containing 5 ml saline solution and thoroughly shaken by a vortex for 1 min. The plasmatreated strains were then spread over either a standard methods agar plate for bacteria culture or a potato-dextrose agar plate for fungi culture. The number of recovered colonies was counted as colony forming units (CFU)/ml after either 3 days of incubation at 37 °C for bacteria growth or 7 days of incubation at 25 °C for fungi growth. In addition to colony counts, scanning electron microscopy (SEM) was performed to obtain detailed information regarding the morphological alterations of the strains of P. aeruginosa and A. niger as previously described [2,5]. The plasma-treated and non-treated silk fabrics were sputter-coated with gold/platinum using an ion coater (E1010, Hitachi, Tokyo, Japan) and observed under an electron microscope (S-800, Hitachi) at an accelerating voltage of 20 kV.
(MTS 858, MTS Systems Co., Eden Prairie, MN). A displacement of 10 mm/min was applied to the fabrics along with the longitudinal direction, and all data were collected at a frequency of 20 Hz until break occurred. 2.4.2. SEM observation The surface morphology of silk fabrics treated without or with microwave-induced argon plasma was observed under a scanning electron microscope (Hitachi S-800) as mentioned above. 2.4.3. Evaluation of color change of silk fabrics using L⁎a⁎b⁎ color coordinate system For evaluating the color changes of silk fabrics, the fabrics was prepared as a disk of 0.9 cm in diameter and 0.2 cm in thickness and treated with plasma for 1–5 s. After plasma treatment, the color change was analyzed by the L⁎a⁎b⁎ [L⁎: lightness (or luminance), a⁎: balance between green (−) and red (+), b⁎: balance between blue (−) and yellow (+)] color coordinate system in combination with a personal computer as previously described [13]. Briefly, the L⁎a⁎b⁎ color difference between the non-treated and plasma-treated fabrics was measured at four different points on each fabric with a central cross stripe connected to a computer. All variables were tested in three independent measurements, which were repeated twice (n = 24). Quantitative data were expressed as mean± standard deviation. Statistical comparisons were carried out with repeated ANOVA (SPSS 12.0 for windows). A value of p b 0.05 was considered statistically significant. 3. Results and discussion 3.1. Sterilization effects of plasma treatment on microorganisms in silk fabrics The sterilization effects of microwave-induced argon plasma at atmospheric pressure on bacterial and fungal strains in silk fabrics are shown in Fig. 1. Although S. aureus was the most resistant bacterial strain to the plasma treatment, all the strains were completely sterilized in less than 7 s. The number of recovered colonies after plasma treatment was significantly decreased in a time-dependent manner. In our previous studies, it has already been shown that several bacterial and fungal strains can be fully sterilized and inactivated by plasma treatment within 20 s or less [2,5,6]. Therefore, it is suggested that microwave-induced argon plasma at atmospheric pressure can sterilize silk fabrics contaminated with microorganisms. Recent studies have reported that the plasma-treated textiles or fabrics showed a significant increase in the incorporation of oxygencontaining groups (e.g., C–O, O–CfO and CfO), which allowed the
2.4. Effects of plasma treatment on physical properties of silk fabrics 2.4.1. Mechanical test In order to determine the influence of plasma treatment on the mechanical properties of silk fabrics, the tensile strength at break of the fabrics was measured before and after plasma treatment for 1–5 s. The dimension of the testing fabrics was prepared equally to that of the fabrics used for sterilization test. According to the guidelines of ASTM D5035-06 [12], the ultimate tensile strength of plasma-treated or non-treated fabrics was determined using a material testing system
Fig. 1. Sterilization effects of microwave-induced argon plasma treatment on silk fabrics inoculated with some kinds of bacterial and fungal strains.
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
surface of them more reactive and hydrophilic [14,15]. It would seem that the formation of hydrophilic oxygen-containing functional groups on the textiles might lead to enhanced microbial growth on the fiber surface over a period of time. However, the problem of recontamination of the fabrics would be resolved if they were kept after plasma treatment at the place of which the environmental conditions, including humidity, temperature and air, were controllable. 3.2. Morphological observations of plasma-treated microorganisms in silk fabrics The sterilization effect of plasma treatment was confirmed by SEM micrographs showing the morphological alterations of the strains of P. aeruginosa (Fig. 2) and A. niger (Fig. 3) by plasma treatment for 5 s. The morphology of the non-treated P. aeruginosa spores was a typically round shape with average dimensions of 0.6 μm in diameter (Fig. 2A and B). In contrast, the plasma-treated spores were completely destroyed with severely damaged and ruptured cell membranes
5775
(Fig. 2C). Moreover, the intracellular contents were all released into the surrounding surface after plasma treatment and the original shape and structure of spores disappeared with microscopic debris (Fig. 2D). Fig. 3 shows the SEM images of A. niger before and after plasma treatment. It was revealed that the non-treated normal spores of A. niger had a globular shape (Fig. 3A and B), while the plasma-treated spores for 5 s were clearly damaged with holes in the cell walls (an insert in Fig. 3C), significant reduction in size as well as a transformed and amorphous morphology (Fig. 3D). It is well known that two combined mechanisms are involved in the plasma-mediated sterilization as follows: the destruction of cell wall and DNA by UV irradiation and activated free radicals, and the erosion of microorganisms through etching eventually enhanced by UV radiation [5,16,17]. The UV and activated free radicals generated during plasma treatment weaken the cell wall of the microorganisms by reacting with the hydrocarbon bonds and cause the disruption of unsaturated bonds, particularly the purine and pyrimidine components of the nucleoproteins. As the process continues, the plasma
Fig. 2. SEM micrographs of morphology of P. aeruginosa in silk fabrics before (A and B) and after (C and D) microwave-induced argon plasma treatment for 5 s.
5776
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
Fig. 3. SEM micrographs of morphology of A. niger in silk fabrics before (A and B) and after (C and D) microwave-induced argon plasma treatment for 5 s.
removes the outer cell wall layer of the microorganisms with its burst and destruction. Another sterilization process of microwave plasma is the erosion of the microorganisms through etching to form volatile compounds through slow combustion by oxygen atoms or radicals emanating from the plasma. 3.3. Effects of plasma treatment on tensile strength and surface morphology of silk fabrics To investigate the effects of microwave-induced argon plasma treatment on the mechanical property of silk fabrics, the tensile strength at break was determined according to the guidelines of ASTM. Mechanical analysis revealed that plasma treatment did not adversely affect the tensile strength of silk fabrics (Fig. 4A). The ultimate tensile strength of the fabrics was slightly decreased by plasma treatment, but there was no significant difference between the non-treated and plasma-treated fabrics. Poll et al. also demonstrated that plasma was able to change the textile surface properties without affecting the interior of the fibers [18]. Microwave-induced plasma by
the atmospheric press gas discharge is a source of electrons, ions, excited atoms and molecules, active free radicals and UV radiation. These factors allow the plasma to function as a unique sterilizing agent for the protection and conservation of various materials, including polymers, papers and fabrics [17]. The major advantage of microwaveinduced plasma treatment to papers or fabrics is the potential of cleaning and sterilization without deterioration of their mechanical strength [19]. The surface morphologies of the non-treated and plasma-treated silk fabrics were then observed by SEM (Fig. 4B–E). It was revealed that plasma treatment did not alter the surface morphology of the fabrics. The plasma-treated fabrics were shown to have almost similar morphology to that of the non-treated fabrics. 3.4. Effects of plasma treatment on color change of silk fabrics To examine the effects of microwave-induced argon plasma treatment on the color change of silk fabrics, the lightness (L⁎), balance (a⁎) between green and red, and balance (b⁎) between blue
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
5777
Fig. 4. Effects of microwave-induced argon plasma treatment on tensile strength and surface morphology of silk fabrics. (A) Change of ultimate tensile strength of the fabrics according to the plasma treatment time. SEM micrographs of the fabrics treated without (B and C) or with (D and E) microwave-induced argon plasma for 5 s.
5778
D.J. Park et al. / Surface & Coatings Technology 202 (2008) 5773–5778
plasma treatment (Fig. 5A). It was also found that the color intensities of green and yellow were significantly (p b 0.05) increased after 4 s of plasma treatment (Fig. 5B and C). It has been already reported that the L⁎a⁎b⁎ color coordinate system was very objective and quantitative and could be useful for the evaluation of treatment effects on colored skin lesions [20]. 4. Conclusions In the present study, it was shown that microwave-induced argon plasma treatment could completely sterilize the silk fabrics contaminated with various strains of microorganisms. It was also found that the plasma treatment did not affect the ultimate tensile strength and surface morphology of the fabrics whereas it decreased the lightness of the fabrics with concomitant increases in the color intensities of green and yellow. These results suggest that microwaveinduced argon plasma can be effectively used as an alternative method for sterilizing and protecting cultural assets out of silk fabrics. Acknowledgements This work was supported by Korea Science and Engineering Foundation (KOSEF, Grant No. R01-2007-000-20472-0). References [1] S.R. Goode, K.W. Baughman, Appl. Spectrosc. 38 (1984) 755. [2] 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. [3] N. Philip, B. Saoudi, M-.C. Crevier, M. Moisan, J. Barbeau, J. Pelletier, IEEE Trans. Plasma Sci. 30 (2002) 1429. [4] A.V. Khomich, I.A. Soloshenko, V.V. Tsiolko, I.L. Mikhno, Proc. of the 12th International Conference on Gas discharges & Their Applications 2 (1997) 740. [5] 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. [6] B.J. Park, K. Takatori, M.H. Lee, D.-W. Han, Y.I. Woo, H.J. Son, J.K. Kim, K.-H. Chung, S.O. Hyun, J.-C. Park, Surf. Coat. Technol. 201 (2007) 5738. [7] A.D. Lucas, K. Merritt, V.M. Hitchins, T.O. Woods, S.G. McNamee, D.B. Lyle, S.A. Brown, J. Biomed. Mater. Res. B Appl. Biomater. 66 (2003) 548. [8] Y. Henon, Med. Device Technol. 3 (1992) 30. [9] T.T. Chau, C.K. Kwan, B. Gregory, M. Fransisco, Biomaterials 17 (1996) 1273. [10] K.-Y. Lee, B.J. Park, D.H. Lee, I.-S. Lee, S.O. Hyun, K.-H. Chung, J.-C. Park, Surf. Coat. Technol. 193 (2005) 35. [11] B.J. Park, K. Takatori, Y. Sugita-Konishi, I.-H. Kim, M.H. Lee, D.-W. Han, K.-H. Chung, S.O. Hyun, J.-C. Park, Surf. Coat. Technol. 201 (2007) 5733. [12] Standard Test Method for Breaking Force and Elongation of Textile Fabrics (Strip Method), ASTM D5035-06 American Society for Testing and Materials, Vol. 07.02 (2000). [13] S.C. Kim, D.W. Kim, J.P. Hong, D.K. Rah, Yonsei Med. J. 41 (2000) 333. [14] R. Morent, N. De Geyter, C. Leys, L. Gengembre, E. Payen, Text. Res. J. 77 (2007) 471. [15] Y.Q. Zhu, E.T. Kang, K.G. Neoh, L. Chan, D.M.Y. Lai, A.C.H. Huan, Appl. Surf. Sci. 225 (2004) 144. [16] M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, M. Tabrizian, L.H. Yahia, Int. J. Pharm. 226 (2001) 1. [17] M. Laroussi, IEEE Trans. Plasma Sci. 30 (2002) 1409. [18] H.U. Poll, U. Schladitz, S. Schreiter, Surf. Coat. Technol. 142/144 (2001) 489. [19] U. Vohrer, I. Trick, J. Bernhardt, C. Oehr, H. Brunner, Surf. Coat. Technol. 142/144 (2001) 1069. [20] D.K. Rah, S.C. Kim, K.H. Lee, B.Y. Park, D.W. Kim, Plast. Reconstr. Surg. 108 (2001) 842.
Fig. 5. Effects of microwave-induced argon plasma treatment on color change of silk fabrics (*p b 0.05 vs. the non-treated, analyzed by repeated ANOVA, n = 24).
and yellow of the fabrics were analyzed using the L⁎a⁎b⁎ color coordinates. It was shown that the lightness of silk fabrics was significantly (p b 0.05) decreased in a time-dependent manner by