Sterilization and Microorganism Deactivation Using Atmospheric Plasma

The following articles, papers and publications on Sterilization and Microorganism Deactivation Using Atmospheric Plasma address key topics such as Escherichia coli deactivation (inactivation of E Coli), biofilm inactivation. This work using atmospheric plasma sterilization can also be related to the sterilization of PCR plates, microarrays, medical devices and foods.

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• Bomi Gweon, D.B. Kim, S.Y. Moon, W. Choe., “Escherichia coli deactivation study controlling the atmospheric pressure plasma discharge conditions.” Current Applied Physics 9 (2009) 625–628. Abstract: Bio-applications of plasma have been widely studied in recent years. However, considering the high interests, the inactivation mechanisms of micro-organisms by plasma have not been clearly explained.The goal of this study was to find the sterilization mechanisms and define the major sterilization factors with the atmospheric pressure radio-frequency helium glow discharge. For the sterilization target the Escherichia coli was used. To begin with the sterilization study, the plasma characteristics were investigated by means of electrical and optical diagnostics. Especially, the gas temperature was controlled under 50 C by keeping the input power less than 70W to eliminate the thermal effects. Contribution of the UV irradiation from the plasma was studied and it turned out to be negligible. On the other hand, it was found that the sterilization was more effective up to 40% with only 0.15% oxygen addition to the helium supply gas. It indicates that the inactivation process was dominantly controlled by oxygen radicals, rather than heat or UV photons.
• Deng S, Ruan R, Mok CK, Huang G, Lin X, Chen P., “Inactivation of Escherichia coli on almonds using nonthermal plasma.” J Food Sci. 2007 Mar;72(2):M62-6. Abstract: This study was carried out to investigate the applicability of nonthermal plasma (NTP) technology for the pasteurization of almonds. Almonds were spiked with various levels of Escherichia coli by dipping the almonds in E. coli culture broth followed by drying. The spiked almonds were treated with NTP under different treatment conditions. The pattern of the microorganisms reduction by NTP was analyzed. NTP was found to be effective on reduction of E. coli on almond evidenced by almost 5-log reduction after 30-sec treatment at 30 kV and 2000 Hz. The NTP bactericidal effect on E. coli inoculated on almond increased with the applied voltage and the frequency. The NTP reduction followed the 1st-order reaction kinetics, and the reduction rate constants varied with almond types and grades. The E. coli cells at logarithmic phase were more sensitive to the NTP than those at stationary and declining phases.
• Hyun Pa Song, Binna Kim, Jun Ho Choe, Samooel Jung, Se Youn Moon, Wonho Choe,Cheorun Jo., “Evaluation of atmospheric pressure plasma to improve the safety of sliced
  cheese and ham inoculated by 3-strain cocktail Listeria monocytogenes.” Food Microbiology 26 (2009) 432–436. Abstract: The objective of this study was to evaluate the efficacy of atmospheric pressure plasma (APP), which is capable of operating at atmospheric pressure in air, in sliced cheese and ham inoculated by 3-strain cocktail of Listeria monocytogenes (ATCC 19114, 19115, and 19111, LMC). The process parameters considered were input power (75, 100, 125, and 150 W) and plasma exposure time (60, 90, and 120 s). Microbial log reduction increased with increases of input power and plasma exposure time. After 120 s APP treatments at 75, 100, and 125 W, the viable cells of LMC were reduced by 1.70, 2.78, and 5.82 log in sliced cheese, respectively. More than 8 log reductions can be achieved in 120 s at 150 W. In contrast, reductions after 120 s ranged from 0.25 to 1.73 log CFU/g in sliced ham. Calculated D values, the exposure time required to inactivate 90% of a population, from the survival curves of 75, 100, 125, and 150Wof APP treatments were 71.43, 62.50, 19.65, and 17.27 s for LMC in sliced cheese, respectively, and those in sliced ham were 476.19, 87.72, 70.92, and 63.69 s. No viable cells were detected at 125 and 150 W of APP treatment in sliced cheese, irrespective of plasma exposure time, after 1 week at a detection limit of 101 CFU/g. These results indicate that the inactivation effects of APP on L. monocytogenes are strongly dependent on the type of food.
• Se Youn Moon, D.B. Kim, B. Gweon, W. Choe, H.P. Song, Cheorun Jo,. “Feasibility study of the sterilization of pork and human skin surfaces by atmospheric pressure plasmas.” Thin Solid Films 517 (2009) 4272–4275. Abstract: Atmospheric pressure radio frequency (rf) glow discharge characteristics were studied aiming for the plasma treatment of living tissues such as pork and human skin. Electrical and optical measurements of the plasma gave reasonable values at low current of 4 mA and gas temperature of 60 °C at 100 W. The colorimetric measurement of the treated pork sample demonstrated negligible thermal effect from the plasma. In addition, the sterilization efficiency of Escherichia coli inoculated on pork surface was comparable with that of a conventional UV sterilizer. The experimental results promise that the atmospheric pressure rf plasma can be safely applied to human skin treatment without electrical and thermal damage.
• Hyejeong Yun, Binna Kim, Samooel Jung, Zbigniew A. Kruk, Dan Bee Kim, Wonho Choe, Cheorun Jo., “Inactivation of Listeria monocytogenes inoculated on disposable plastic tray,aluminum foil, and paper cup by atmospheric pressure plasma.” Food Control 21 (2010) 1182–1186. Abstract: The objective of this study was to investigate the effect of atmospheric pressure plasma (APP) on Listeria monocytogenes inoculated onto disposable food containers including disposable plastic trays, aluminum foil, and paper cups. The parameters considered in APP processing were input power (75, 100, 125, and 150 W) and exposure time (60, 90, and 120 s). The bacterial reduction in the disposable plastic trays, aluminum foil, and paper cups was associated with increased input power and exposure time of APP. The D10 values were calculated as 49.3, 47.7, 36.2, and 17.9 s in disposable plastic trays, 133, 111, 76.9, and 31.6 s in aluminum foil and 526, 65.8, 51.8, and 41.7 s in paper cups at 75, 100, 125, and 150Wof input power,
  respectively. There were no viable cells detected after 90 and 120 s of APP treatment at 150W in disposable plastic trays. However, only three decimal reductions of viable cells were achieved in aluminum foil and paper cups at 150W for 120 s. These results demonstrate that APP treatment is effective for inactivation of L. monocytogenes and applicable for disposable food containers. However, the type of material is crucial and appropriate treatment conditions should be considered for achieving satisfactory inactivation level.
• Joaquin, J. C., Kwan, C., Abramzon, N., Vandervoort, K., and Brelles-Marino, G., “Is gas-discharge plasma a new solution to the old problem of biofilm inactivation?” Microbiology 155, 724 (2009). Abstract: Conventional disinfection and sterilization methods are often ineffective with biofilms, which are ubiquitous, hard-to-destroy microbial communities embedded in a matrix mostly composed of exopolysaccharides. The use of gas-discharge plasmas represents an alternative method, since plasmas contain a mixture of charged particles, chemically reactive species and UV radiation, whose decontamination potential for free-living, planktonic micro-organisms is well established. In this study, biofilms were produced using Chromobacterium violaceum, a Gram-negative bacterium present in soil and water and used in this study as a model organism. Biofilms were subjected to an atmospheric pressure plasma jet for different exposure times. Our results show that 99.6 % of culturable cells are inactivated after a 5 min treatment. The survivor curve shows double-slope kinetics with a rapid initial decline in c.f.u. ml(-1) followed by a much slower decline with D values that are longer than those for the inactivation of planktonic organisms, suggesting a more complex inactivation mechanism for biofilms. DNA and ATP determinations together with atomic force microscopy and fluorescence microscopy show that non-culturable cells are still alive after short plasma exposure times. These results indicate the potential of plasma for biofilm inactivation and suggest that cells go through a sequential set of physiological and morphological changes before inactivation.
• Hong, Y. F., Kang, J. G., Lee, H, Y., Uhm, H. S., Moon, E., and Park, Y. H., ” Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores,” Lett. Appl. Microbiol. 48, 33 (2009). Abstract: AIMS: Escherichia coli and Bacillus subtilis spores were treated with an atmospheric plasma mixture created by the ionization of helium and oxygen to investigate the inactivation efficiency of a low-temperature plasma below 70 degrees C. METHODS AND RESULTS: An electrical discharge plasma was produced at a radio frequency (RF) of 13.56 MHz, connected to a perforated circular electrode with a discharge spacing of 1-15 mm. The discharge gas was helium with 0-2% oxygen. For the plasma treatment, a dried E. coli cell or B. subtilis endospore suspension on a cover-glass was exposed to oxygen downstream of the plasma from holes in an RF-powered electrode. The sterilization effect of the RF plasma was highest with 0.2% oxygen, corresponding to the maximum production of oxygen radicals. CONCLUSIONS:
  Oxygen radicals generated by RF plasma are effective for the destruction of bacterial cells and endospores. SIGNIFICANCE AND IMPACT OF THE STUDY: Low-temperature atmospheric plasma can be used for the disinfection of diverse objects, especially for the inactivation of bacterial endospores
• Simon, A., Anghel, S. D., Papiu, M., and Dinu, O., “Diagnostics and active species formation in an atmospheric pressure helium sterilization plasma source,” Nucl. Instrum. Methods Phys. Res., Sect. B 267, 438 (2009). Abstract: Systematic spectroscopic studies and diagnostics of an atmospheric pressure radiofrequency (13.56 MHz) He plasma is presented. The discharge is an intrinsic part of the resonant circuit of the radiofrequency oscillator and was obtained using a monoelectrode type torch, at various gas flow-rates (0.1–6.0 l/min) and power levels (0–2 W). As function of He flow-rate and power the discharge has three developing stages: point-like plasma, spherical plasma and ellipsoidal plasma. The emission spectra of the plasma were recorded and investigated as function of developing stages, flow-rates and plasma power. The most important atomic and molecular components were identified and their evolution was studied as function of He flow-rate and plasma power towards understanding basic mechanisms occurring in this type of plasma. The characteristic temperatures (vibrational Tvibr, rotational Trot and excitation Texc) and the electron number density (ne) were determined.
• Li, S. Z., and Lim, J. P., ” Comparison of sterilizing effect of nonequilibrium atmospheric-pressure He/O2 and Ar/O2 plasma jets,” Plasma Sources Sci. Technol. 10, 61 (2008). Abstract: The sterilizing effect of the non-equilibrium atmospheric pressure plasma jet by applying it to the Bacillus subtilis spores is invesigated. A stable glow discharge in argon or helium gas fed with active gas (oxygen), was generated in the coaxial cylindrical reactor powered by the radio-frequency power supply at atmospheric pressure. The experimental results indicated that the efficiency of killing spores by making use of an Ar/O2 plasma jet was much better than with a He/O2 plasma jet. The decimal reduction value of Ar/O2 and He/O2 plasma jets under the same experimental conditions was 4.5 seconds and 125 seconds, respectively. It was found that there exists an optimum oxygen concentration for a certain input power, at which the sterilization efficiency reaches a maximum value. It is believed that the oxygen radicals are generated most efficiently under this optimum condition.
• Kolb, J. F., Mohamed, A. A. H., Price, R. O., Swanson, R. J., Bowman, A., Chiavarini, R. L., Stacey, M., and Schoenbach, K. H., “Cold atmospheric pressure air plasma jet for medical applications,” J. Appl. Phys. Lett. 92, 241501 (2008). Abstract: By flowing atmospheric pressure air through a direct current powered microhollow cathodedischarge, we were able to generate a 2cm long plasma jet. With increasing flow rate, the flow becomes turbulent and temperatures of the jet are reduced to values close to room temperature. Utilizing the jet, yeast grown on agar can be eradicated with a treatment of only a few seconds. Conversely, animal studies show no skin damage even with exposures ten times longer than needed for pathogen extermination. This cold plasma jet provides an effective mode of treatment for yeast infections of the skin.
• Vandervoort, K. G., Abramzon, N., and Brelles-Marino, G., “Plasma interactions with bacterial biofilms as visualized through atomic force microscopy plasma science,” IEEE Trans. Plasma Sci. 36, 1296 (2008). Bacterial biofilms are microbial communities that are less susceptible to standard killing methods than free-living bacteria. Gas-discharge plasmas were used to treat biofilms for various exposure times. After 5-min plasma exposure, 90% of culturable cells were removed. Atomic-force-microscope images that reveal the sequential changes in cell morphology occurring during plasma treatment are presented.
• Weltmann, K. D., Brandenburg, R. Von., Woedtke, T., Ehlbeck, J., Foest, R., Stieber, M., and Kindel, E., “Antimicrobial treatment of heat sensitive products by miniaturized atmospheric pressure plasma jets (APPJs),” J. Phys. D: Appl. Phys. 41, 194008 (2008). Abstract: The technological potential of non-thermal plasmas for the antimicrobial treatment of heat sensitive materials is well known. Despite a multitude of scientific activities with considerable progress within the last few years, the realization of industrial plasma-based decontamination or sterilization technology remains a great challenge. This may be due to the fact that an antimicrobial treatment process needs to consider all properties of the product to be treated as well as the requirements of the complete procedure, e.g. a reprocessing cycle of medical instruments. The aim of this work is to demonstrate the applicability of plasma-based processes for the antimicrobial treatment on selected heat sensitive products. The strategy is to use modular, selective and miniaturized plasma sources, which are driven at atmospheric pressure and adaptable to the products to be treated. Antimicrobial treatment of heat sensitive products by miniaturized atmospheric pressure plasma jets (APPJs).
• Perez-Martinez, J. A., Pena-Eguiluz, R., Lopez-Callejas, R., Mercado-Cabrera, A., Valencia, R. A., Barocio, S. R., Benitez-Read, J. S., and Pacheco-Sotelo, J. O., ” An RF microplasma facility development for medical applications,” Surf. Coat. Technol. 201, 5684 (2007). Abstract: Microplasmas represent, by their physical nature, a considerable potential for medical applications given their highly accurate action and extremely controllable penetration on the surface of biological tissue. This plasma modality is today a powerful tool in practical applications such as the elimination of necrotic cells or the sterilization of dental cavities. As we start up a research line into this technology, we have constructed a plasma needle capable of producing non thermal plasmas, within a 1 mm radius, produced by a typical 13.56 MHz RF generator. The plasma is developed out of helium and argon. Initial electrical tests show that the plasma needle can operate at comparatively low voltages (peak to peak 100–250 V) and low power consumption. With a view to optimizing the medical applicability of the system, a study of the effects of plasma temperature and power variation at different distances is presented.
• Deng, X. T., Shi, J. J., Chen, H. L., and Kong, M. G., ” Protein destruction by atmospheric pressure glow discharges,” Appl. Phys. Lett. 90, 051504 (2007). Abstract: It is well established that atmospheric pressureglow discharges are capable of bacterial inactivation. Much less known is their ability to destruct infectious proteins, even though surgical instruments are often contaminated by both bacteria and proteinaceous matters. In this letter, the authors present a study of protein destruction using a low-temperature atmospheric dielectric-barrier discharge jet. Clear evidences of protein removal are presented with data of several complimentary experiments using scanning electron microscopy, electron dispersive x-ray analysis, electrophoresis, laser-induced fluorescence microscopy, and protein reduction kinetics. Considerable degradation is observed of protein fragments that remain on their substrate surface after plasma treatment.
• Abramzon, N., Joaquin, J. C., Bray, J., and Brelles-Marino, G., “Biofilm Destruction by RF High-Pressure Cold Plasma Jet Plasma Science,” IEEE Trans. Plasma. Sci. 34, 1304 (2006). Abstract: Biofilms are bacterial communities embedded in a glue-like matrix mostly composed of exopolysaccharides and a small amount of proteins and nucleic acids. Conventional disinfection and sterilization methods are often ineffective with the biofilms since microorganisms within the biofilm show different properties from those in free planktonic life. The use of the gas discharge plasmas is a novel alternative since the plasmas contain a mixture of charged particles, chemically reactive species, and UV radiation. The four-day-old single-species biofilms were produced using Chromobacterium violaceum, a gram-negative bacterium commonly present in soil and water. The gas discharge plasma was produced by using an Atomflo 250 reactor (Surfx Technologies), and the bacterial biofilms were exposed to it for different periods of time. Our results show that a 10-min plasma treatment is able to kill almost 100% of the cells. The results show a rapid initial decline in the colony forming units per milliliter (phase one) that is followed by a much slower subsequent decline (phase two) of the D-values that are longer than the inactivation of the planktonic organisms, suggesting a more complex inactivation mechanism for the biofilms. Two hypotheses are offered to explain this biphasic behavior. Optical emission spectroscopy was used to study the plasma composition, and the role of the active species is discussed. These results indicate the potential of plasma as an alternative way for biofilm removal.
• Gaunt, L. F., Beggs, C. B., and Georghiou, G. E., “Bactericidal action of the reactive species produced by gas-discharge nonthermal plasma at atmospheric pressure: A review,” IEEE Trans. Plasma. Sci. 34, 1257 (2006). Abstract: Biological decontamination using a nonthermal gas discharge at atmospheric pressure in air is the subject of significant research effort at this time. The mechanism for bacterial deactivation undergoes a lot of speculation, particularly with regard to the role of ions and reactive gas species. Two mechanisms have been proposed: electrostatic disruption of cell membranes and lethal oxidation of membrane or cytoplasmic components. Results show that death is accompanied by cell lysis and fragmentation in Gram-negative bacteria but not Gram-positive species, although cytoplasmic leakage is generally observed. Gas discharges can be a source of charged particles, ions, reactive gas species, radicals, and radiation (ultraviolet, infrared, and visible), many of which have documented biocidal properties. The individual roles played by these in decontamination are not well understood or quantified. However, the reactions of some species with biomolecules are documented otherwise in the literature. Oxidative stress is relatively well studied, and it is likely that exposure to gas discharges in air causes extreme oxidative challenge. In this paper, a review is presented of the major reactive species generated by nonthermal plasma at atmospheric pressure and the known reactions of these with biological molecules. Understanding these mechanisms becomes increasingly important as plasma-based decontamination and sterilization devices come closer to a wide-scale application in medical, healthcare, food processing, and air purification applications. Approaches are proposed to elucidate the relative importance of reactive species.
• Goree, J., Liu, B., Drake, D., and Stoffels, E., “Killing of S-mutans bacteria using a plasma needle at atmospheric pressure,” IEEE Trans. Plasma. Sci. 34, 1317 (2006). Abstract: Streptococcus mutans (S. mutans) bacteria were killed using a low-power millimeter-size atmospheric-pressure glow-discharge plasma or plasma needle. The plasma was applied to a culture of S. mutans that was plated onto the surface of an agar nutrient in a Petri dish. S. mutans is the most important microor-ganism for causing dental caries. A spatially resolved biological diagnostic of the plasma is introduced, where the spatial pattern of bacterial colonies in the sample was imaged after plasma treat-ment and incubation. For low-power conditions that would be at-tractive for dentistry, images from this biological diagnostic reveal that S. mutans was killed within a solid circle with a 5-mm di-ameter, demonstrating that site-specific treatment is possible. For other conditions, which are of interest for understanding plasma transport, images show that bacteria were killed with a ring-shaped spatial pattern. This ring pattern coincides with a similar ring in the spatial distribution of energetic electrons, as revealed by Abel-inverted images of the glow. The presence of the radicals OH and O was verified using optical-emission spectroscopy. Index Terms—Atmospheric glow discharge, disinfection, mi-croorganisms, microplasma, nonthermal plasma, plasma applica-tions, sterilization. I.
• Xu, L., Liu, P., Zhan, R. J., Wen, X. H., Ding, L. L., and Nagatsu, M., “Experimental study and sterilizing application of atmospheric pressure plasmas,” Thin Solid Films 400, 506 (2006). Abstract: The atmospheric pressure surface barrier discharge (APSBD) in air has been used in killing Escherichia coli. We have developed two similar dielectric barrier discharge (DBD) structure types of nonthermal plasma jets (PJ) driven by 5–20 kHz audio-frequency power at atmospheric pressure. At a flow rate of 200 L/h (argon), a stable, arc-free discharge was produced. At 1.5 cm from the nozzle, the gas temperature was kept at 47 °C for PJ-1 source and 38 °C for PJ-2 source. Some research on sterilization has been carried out and results show that such a plasma jet source as PJ-2 is very effective in the disruption of E. coli.
• Chu, P. K., Chen, J. Y., Wang, L. P., and Huang, N., “Plasma-surface modification of biomaterials,” Mater. Sci. Eng. Rep. 36, 143 (2002). Abstract: Plasma-surface modification (PSM) is an effective and economical surface treatment technique for many materials and of growing interests in biomedical engineering. This article reviews the various common plasma techniques and experimental methods as applied to biomedical materials research, such as plasma sputtering and etching, plasma implantation, plasma deposition, plasma polymerization, laser plasma deposition, plasma spraying, and so on. The unique advantage of plasma modification is that the surface properties and biocompatibility can be enhanced selectively while the bulk attributes of the materials remain unchanged. Existing materials can, thus, be used and needs for new classes of materials may be obviated thereby shortening the time to develop novel and better biomedical devices. Recent work has spurred a number of very interesting applications in the biomedical field. This review article concentrates upon the current status of these techniques, new applications, and achievements pertaining to biomedical materials research. Examples described include hard tissue replacements, blood contacting prostheses, ophthalmic devices, and other products.

Contact us to receive a complimentary copy of the technical publications on Sterilization and Microorganism Deactivation Using Atmospheric Plasma above. Please reference the authors and publication number in your email request (copy and paste into your email response).