Scanning Electron Microscopy Image of Escherichia Coli Exposed with Atmospheric-Pressure Plasma Jet

The morphological change of Escherichia coli (E. coli) exposed to an atmospheric-pressure argon (Ar) plasma jet was observed by scanning electron microscopy (SEM). The Ar plasma jet was generated at a frequency of 9 kHz, applied voltage of 10 kV, and Ar gas flow rate of 10 L/min at atmospheric pressure. E. coli seeded on an agar plate in a Petri dish was disinfected by Ar plasma jet exposure for 2 seconds. SEM images showed that E. coli cells deformed and faded after the Ar plasma jet exposure. The morphological changes were probably due to the destruction of the cell wall and cell membrane of E. coli caused by the collision of Ar ions and/or electrons in the Ar plasma jet. [DOI: 10.1380/ejssnt.2010.74]


I. INTRODUCTION
Plasma sterilization is an alternative to conventional sterilization methods such as autoclaving, the use of chemicals such as ethylene oxide, gamma ray irradiation, and ultraviolet (UV) light irradiation [1,2].The sterilization and the disinfection of bacteria and fungi using a microwave (2.45 GHz)-induced plasma in the form of a jet at atmospheric pressure have been investigated [3][4][5][6][7].The atmospheric-pressure plasma jet dose not need expensive vacuum systems and pin-point exposure is possible.Ohyama, one of the authors, developed a generator for a nonequilibrium plasma jet of helium (He) and argon (Ar) at atmospheric pressure [8,9].The plasma jet is generated by a dielectric barrier discharge using a lowfrequency (9 kHz) high-voltage power source.The temperature of the Ar plasma jet measured using a K-type (chromel-alumel) thermocouple thermometer was about 30 • C [10].
In this work, we present the disinfection of Escherichia coli (E.coli) using the low-temperature Ar plasma jet developed by Ohyama.E. coli seeded on an agar plate in a Petri dish was exposed to the Ar plasma jet.In addition, the morphological change of E. coli after Ar plasma jet exposure was observed by scanning electron microscopy (SEM).

II. EXPERIMENTAL
The schematic configuration of the atmosphericpressure Ar plasma jet generator is shown in Fig. 1.The electrode system was a coaxial cylinder attached to the inner and outer surfaces of a quartz tube.The quartz tube  had an inner diameter of 6 mm, a thickness of 0.5 mm and a length of 100 mm.The inner discharge electrode was a copper tube with a thickness of 0.5 mm and an inner diameter of 4 mm, connected to a high-voltage power source.In addition, the inner electrode was used as an injector of Ar gas for plasma generation.The outer electrode was a copper tube with a thickness of 0.05 mm and a length of 10 mm, connected to the ground via the measurement circuit.The gas gap distance between these electrodes was fixed to 1 mm along the direction of the dielectric barrier surface.In this experiment, the plasma was made of Ar gas at a gas flow rate of 10 L/min.A high-voltage power source was used at a frequency of 9 kHz and an applied voltage of 10 kV.Under the above conditions, the length of the Ar plasma jet from the quartz tube nozzle exit was approximately 3 cm, as shown in Fig. 2. E. coli was seeded at 10 6 cells per dish on a 3-mm-thick agar plate in a 90-mm-diameter Petri dish, as shown in Fig. 3. E. coli cells within the "O" at the center of the Petri dish were exposed to the Ar plasma jet.The exposure time was set to 2 and 5 seconds.The distance from the quartz tube nozzle exit to the surface of the agar plate was 2 mm.After Ar plasma jet exposure, samples were incubated at 37 • C for 24 hours.The morphological change of E. coli after Ar plasma jet exposure was observed by SEM (JEOL, JSM-840A).

III. RESULTS AND DISCUSSION
Figure 4 shows photographs of samples incubated for 24 hours after Ar plasma jet exposure for 2 and 5 seconds.For both samples, no E. coli colony was observed in the area exposed to the Ar plasma jet, while E. coli colonies were observed in the areas not exposed to the Ar plasma jet on the agar plate.This result indicates that Ar plasma jet exposure is very useful for the disinfection of E. coli.E. coli seeded on the agar plate in the Petri dish were disinfected with less than 2 seconds of exposure.The diameters of the disinfected area were about 7 and 10 mm with exposure times of 2 and 5 seconds, respectively.The viable cell counts of E. coli exposed to the plasma jet for 2 and 5 seconds were determined by the colony count method and found to be 0 for these exposure times.
The disinfection of E. coli using an Ar plasma in the form of a jet at atmospheric pressure was investigated by Park et al. [5].E. coli seeded on an agar was disinfected within 20 seconds.Sato et al. reported the disinfection efficacy of a coaxial microwave (2.45 GHz) Ar plasma flow at atmospheric pressure against E. coli filtered onto membrane filters [6].They found that E. coli was disinfected by exposure to the plasma flow for 2 min.The disinfection effects of an atmospheric brush-shaped plasma against E. coli seeded in a filter-paper medium were investigated by Yu et al. [7].They found that E. coli was disinfected by exposure to the plasma for about 2 min.The disinfection time (2 seconds) obtained by us is considerably shorter than the above disinfection times.It is considered that the short exposure distance is the reason for realizing a short exposure time in our method com- pared with conventional methods.For microwave-induced plasma, the exposure distance was as long as 124 mm [6].
It is considered that the distance is set long to suppress the disinfection by heat due to the high temperature of microwave-induced plasma.In this case, E. coli is not directly exposed to the plasma because of the long exposure distance.In contrast, in our study, the exposure distance can be set to be as small as 2 mm because of the low temperature of the plasma jet (approximately 30 • C); therefore, E. coli is directly exposed to the plasma.We considered that this difference enables the disinfection of E. coli in a shorter period in our study than in conventional methods.
The morphological change of E. coli after Ar plasma jet exposure was observed by SEM. Figure 5 shows the SEM images of E. coli.Initially, E. coli cells were about 2 µm long and about 0.5 µm in diameter, as shown in Fig. 5(a).After Ar plasma jet exposure for 5 seconds, the shape was deformed and the cell wall was faded, as shown in Fig. 5(b).Although several SEM images of bacteria such as E. coli and fungi exposed with plasmas have been reported [1][2][3][7][8][9], there is no SEM image like our image.This morphological change suggests the destruction of the cell wall and cell membrane of E. coli.Sato et al. found that potassium leaks from E. coli cells after plasma flow exposure and reported the destruction of the cell membrane and cell wall on the basis of their findings.[6].Therefore, the disinfection effects of the atmospheric-pressure Ar plasma jet are probably due to the physical damage of the cell wall and cell membrane caused by the collision of Ar ions and/or electrons in the Ar plasma jet.

IV. CONCLUSIONS
E. coli seeded on an agar plate in a Petri dish were exposed to an Ar plasma jet generated at a frequency of 9 kHz and applied voltage of 10 kV, with an Ar gas flow rate of 10 L/min at atmospheric pressure.After 2 and 5 seconds of Ar plasma jet exposure, no E. coli colony was observed in the exposure area on the agar plate.This result indicates that E. coli is disinfected in less than 2 seconds.The results of SEM observation showed that E. coli cells were deformed and indistinct after Ar plasma jet exposure.Therefore, the disinfection effects of an Ar plasma jet against E. coli are probably due to the physical damage of the cell wall and cell membrane caused by the collision of charged particles such as Ar ions and/or electrons in the Ar plasma jet.

FIG. 3 :
FIG. 3: Photograph of seeded E. coli.Ar plasma jet was exposed within the "O" in the Petri dish.