22G11
Plasma-spore of P. digitatum Interactions in Low Temperature AtmosphericPressure Plasma
低温大気圧プラズマとミドリカビ胞子の相互作用	 
Hiroshi Hashizume1, Takayuki Ohta1, Takumi Mori2, Masafumi Ito1, Keigo Takeda3,Kenji Ishikawa3 and Masaru Hori3
橋爪博司1, 太田貴之1，森	  拓海2，伊藤昌文1, 竹田圭吾3, 石川健治3, 堀	  勝3
1Faculty of Science and Technology, Meijo University, 1-501, Shiogamaguchi, Tempaku-ku, Nagoya 468-8502, Japan2
Faculty of Systems Engineering, Wakayama University, 930, Sakaedani, Wakayama 640-8510, Japan3
Departmanent of Electrical Engineering and Computer Science, Nagoya University,Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan1
名城大学理工学部 〒468-8502	  名古屋市天白区塩釜口1-501
2
和歌山大学システム工学部 〒640-8510	  和歌山市栄谷930
3名古屋大学大学院電子情報システム専攻 〒464-8603	  名古屋市千種区不老町
The spores were inactivated using an oxygen-radical source that supplies only neutral oxygen radicals. Theinactivation rate of Penicillium digitatum spores was correlated with the ground-state atomic oxygen [O(3Pj)]. The result indicates that O (3Pj) is the dominant species in the inactivation. The effect of plasma
treatment on cell membrane of the spores of P. digitatum was observed by using fluorescent microscopy
with 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarboeyanine perchlorate (DiI). The analysis revealed thatplasma treatment caused the staining of intracellular organelles, in addition to cell membrane.

1. Introduction
Methods of inactivating microorganisms using
a non-equilibrium plasma processing haverecentlyattracted
muchattention.
Non-equilibrium plasma processing has many
advantages with respect to low-temperatureprocessing and short processing times. Plasma
inactivation has proved to be an effective systemthat causes minimal damages to the instruments.
Many factors which can possibly cause
inactivation, for example vacuum ultlaviolet(VUV) and UV-C emissions, neutral and charged
species, electric fields, and their synergisticeffects, have been studied [1]. It is reported thatreactive oxygen species (ROS) including
ground-state atomic oxygen [O (3Pj=0,1,2)],hydroxyl radicals (OH), metastable oxygen
molecules (1O 2), and superoxide anions (O 2-) are
effective in inactivating bacteria such as E. coliand Bacillus. Other reports are shown that the O orOH radical due to oxidation-decomposition of cell
membrane is important factor for the inactivation.
[2, 3] However, few studies have investigated therole of O (3Pj) based on quantitative analysis of
the gas phase.
We reported that the spores of Penicillium
digitatum were rapidly inactivated by high-density
non-equilibrium atmospheric pressure plasma(NEAPP) [4]. We investigated the inactivation
effects of ozone and UV radiation. These results
showed that the inactivation rate of this plasma wasnearly one-thousandth of that of an ozonizer using
the integrated number density of ozone measured
by UV absorption spectroscopy and thecontribution of UV radiation toward inactivation
was not dominant for P. digitatum inactivation byNEAPP.
In this study, we investigated the inactivation ofthe spores by using an oxygen radical source that
employs high-density atmospheric-pressure O2/Ar
plasma. The O (3Pj) density was measured using
VUV absorption spectroscopy. The inactivation
efficiency of O (3Pj) is discussed in terms of theatomic oxygen densities estimated by quantitative
analysis of the gas phase. Furthermore, in order toinvestigate the change of the spores of P. digitatum
treated by plasma, we also observed the spores byfluorescent microscopy.
2. Experiments
The radical source was developed based on thehigh-density NEAPP. The NEAPP generates
high-density electrons of about 1016 cm-3. [5, 6] Theabsolute density of O (3Pj) was measured by VUVabsorption spectroscopy using a microdischarge
hollow-cathode lamp (MHCL). Measurements andexposure of radicals to spores were performed 10mm downstream from the radical source. The
inactivation efficiency of the spores was evaluatedby colony-counting method.
Fluorescence image was observed by using aninverted microscope (IX 70 by Olympus) withcharge-coupled device camera (DP 72 by Olympus).
1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) was used as a vitalfluorescence membrane dye [7]. Octadecyl group ofDiI binds acyl group in the phospholipid of the cell
membrane, so that the membrane is strained.
3. Results and Discussion
Figure 1 shows O (3Pj) densities and D values,
which represent indicator of inactivation, as afunction of the O2/(Ar+O2) flow rate ratio. The O
densities were measured to be from 1.4x1014 to
1.5x1015 cm-3. The O density increased withincreasing O2/(Ar+O2) mixture flow rate ratio up to
0.6% and it then decreased with increasing flowrate ratio. On the other hand, the D value decreased
with increasing O2/(Ar+O2) mixture flow rate ratio
up to 0.6% and increased with increasing flow rateratio. Since the D value is in inverse relation to theinactivation rate, O (3Pj) is the dominant species inthe inactivation.
In order to investigate the change of spores of P.
digitatum by treatment of plasma, we observed thespores by fluorescent staining. Figure 2 shows
fluorescence microscopic images of the spore
stained by DiI. In the control, the membrane of thespore was stained by DiI (Fig. 2(a)). On the other
hand, when the spore was treated by the plasma, theintracellular organelles of some spores emitted thefluorescent light by DiI (Fig. 2(b)). For the living
cell, the DiI is not permeable because themembrane has selective permeability. Thus, it issuggested that the DiI penetrated the spore insidethrough the cell membrane. Therefore, it wasspeculated that the membrane was decomposed by
the O or OH radicals produced from the plasma.

Fig. 1 O (3Pj) densities and D values as a function ofO2/(O2+Ar) flow rate ratio.

(a) control(b) plasma treatment
Fig. 2 Fluorescent images of spores of P. digitatum.

4. Conclusion	  We have inactivated P. digitatum spores using
neutral ground-state atomic oxygen generated by anoxygen radical source employing a non-equilibrium
atmospheric-pressure remote O2/Ar plasma. The
ground-state oxygen radical O (3Pj) densities weremeasured using VUV absorption spectroscopy. The
inactivation rate of P. digitatum spores inversely
correlated to the O (3Pj) densities. Accordingly, O(3Pj) is one of the dominant species responsible forinactivating microorganisms. We also observed thespores of P. digitatum by using fluorescent
microscopy using DiI in order to investigate theeffect of oxidation. These results indicated that thecell membrane was destroyed by reactive species
produced from the plasma due to theoxidation-decomposition.
5. Acknowledgment	  This work was partly supported by theKnowledge Cluster Initiative (Second Stage), TokaiRegion Nanotechnology Manufacturing Cluster,and a Grant-in-Aid for Scientific Research on
Innovative Areas, “Frontier Science of Interactions
between Plasmas and Nano-interfaces” (No.
21110006) from the Ministry of Education, Culture,Sports, Science and Technology of Japan.
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