23P019-O
Effect of Atmospheric Plasma Irradiation to Yeast Fungus and Colon Bacillus
酵母および大腸菌への大気圧プラズマ照射の影響
Yoshihito Yagyu, Shouta Sakamoto, Minako Noda, Takashi Yamasaki, Nobuya Hayashi,
Hiroharu Kawasaki, Tamiko Ohshima, Yoshiaki Suda
柳生義人，坂本翔太，野田美菜子，山崎隆志，林信哉，川崎仁晴，大島多美子，須田義昭
Sasebo National College of Technology
1-1 Okishin-cho Sasebo, Nagasaki 857-1193, Japan
佐世保工業高等専門学校 〒857-1193 長崎県佐世保市沖新町1-1
Atmospheric plasma has taken an important part for practical applications for plasma sterilization. Plasma
jet and surface discharge as typical atmospheric plasma source were applied to eliminate microorganisms
in the study. No colonies around the point of exposing plasma jet and surface discharge were observed, andthe area of it tends to depend on irradiation period and species of microorganism. It was supposed thatsome ambient molecules were converted into active species for sterilization agent by atmospheric plasma
such as ozone, singlet oxygen molecule and nitrogen oxide. Furthermore, inhibition area of forming colonytended to become larger with irradiation of surface discharge in comparison with plasma jet.

1. Introduction
Active species generated in plasma have been
become a major focus for sterilization as
substitution of practical sterilization method
using high pressure steam (autoclave sterilization) and ethylene oxide gas (EOG sterilization,C2H4O) [1]. Simultaneously, mechanism andnovel application of plasma sterilization havebeen also studied actively in a decade [2][3].
Phenomena of plasma irradiation to biologicalobjects, such as living body, fruits and vegetables,
have become one of the major topics. Atmospheric plasma has taken an important part forpractical applications because a target of plasma
sterilization has difficulties to keep characteristics under low pressure. Plasma jet and surface
discharge as typical atmospheric plasma source
were applied to eliminate microorganisms andeffect of atmospheric plasma irradiation isreported in this study.
2. Experimental instruction
Atmospheric plasma equipments are applied tothis study, and AC high voltage power unit (Logyelectric Co., LHV-10AC) rated 10kV with 9kHz
to 11kHz is prepared as stable source of power.
Typical plasma jet equipment shown in Fig. 1 andsurface discharge element shown in Fig. 2 wereapplied to this study. Plasma jet was generated
with an electrode placed around φ5mm glass tube.
Helium gas flow of each glass tubes was kept on
0.5L/min consistently. Surface discharge element
made of ceramic plate (37mm long × 15mm wide× 0.4mm thick) placed between electrodes madeof evaporated tungsten. Anode has 22mm long ×6mm wide with 100µm thick and cathode has

30mm long × 1mm wide with 100µm thick.
Surface discharge element was arranged parallelin four lines. Irradiation period of atmosphericplasma is varied from 0min to 10min.
Two species of microorganisms were prepared
for irradiation of atmospheric plasma. Yeast fungus(Saccharomyces cerevisiae, NBRC No.2347) andcolon bacillus (Escherichia coli，NBRC No.3972)
were inoculated into liquid culture medium andapplied to plasma treatment as object of the study.

(a) Before ignition(b) After ignition
Fig. 1. Atmospheric helium plasma jet
Cathode electrode

0.4mm

30mm
Cathodeelectrode

Anode electrode

1mm6mm

15mm37mm(a) Details of surface discharge element(b) Surface discharge element arranged
parallel to each otherFig. 2. Surface discharge element

3. Experimental results
3.1 Plasma jet
Typical cultural medium sheets of yeast fungus
and colon bacillus after exposing plasma jet areindicated in Fig. 3. It is found that ring of inhibition
around the point of exposing plasma jet and thediameter of it tends to depend on irradiation period
and species of microorganism. Yeast fungus tends
to have tolerance against plasma exposure incomparison with colon bacillus because eachmicroorganism has different structure of cell walls,
and reactions of plasma to cell walls are supposed
to depend on the species. Plasma jet with heliumgas was exposed into air and it involved especiallyoxygen or nitrogen. Therefore, some ambient
molecules convert into active species forsterilization agent such as ozone, singlet oxygen
molecule and nitrogen oxide.

Physiological saline was applied to cultural sheet
medium instead of 1ml sample solution which waspure water. Inhibition ring of colon bacillus withphysiological saline tended to be larger thanapplying pure water although yeast fungus is almost
same irrespective of sample solution (Fig. 4). It is
supposed that sterilization agents, such as ClO-,
HClO and HClO2, were generated when plasma jet
emitted to physiological saline.
3.2 Surface discharge
Surface discharge was irradiated to yeast fungus
when surface discharge elements close to cultural
medium sheet. It was clearly found that colonies did
not form near surface discharge and inhibition area
tended to increase with duration of irradiation oftreatment. Inhibition area of forming colony
became larger with irradiation of surface discharge
compared to plasma jet. However, effective area ofplasma sterilization should be larger for practicalusage.
18
Radius of inhibition ring ±SE [mm]Effect of atmospheric plasma irradiation wasevaluated with commercially available cultural
medium sheet named Sanita-kun (Chisso Corp.) for“coliform group” and “yeast & mold (Rapid Type)”.
Active microorganism grows and forms colony on
the surface layer of cultural medium sheet, and thecolony of microorganism colors with chromogenic
reaction. Yeast fungus and colon bacillus in liquid
culture medium, whose initial number was adjusted
in 4×103 CFU/ml, were applied onto cultural sheet
medium with 1ml sample solution, and thenatmospheric plasma was irradiated near the centreof cultural sheet medium. After exposing
atmospheric plasma to microorganisms, cultural
sheet medium for “coliform group” and “yeast &mold (Rapid Type)” were incubated at 35°C for 24hours and 25°C for 48 hours, respectively.

Colon bacillus[w/ pure water]
Colon bacillus[w/ saline]
Yeast fungus[w/ pure water]
Yeast fungus[w/ saline]

16141210864200246Irradiation period [min]810Fig. 4 Relationship between irradiation period ofplasma jet and radius of inhibition ring.

0min(cont.)
2min
5min
Fig. 5 Cultural medium sheets after Surface
discharge irradiation
0min(cont.)
5min
10min
(a) Yeast fungus, Saccharomyces cerevisiae

0min(cont.)
5min
10min
(b) Colon bacillus, Escherichia coli
Fig. 3 Cultural medium sheets (Sanita-kun,Chisso Corp.)after plasma jet irradiation

Acknowledgments
This study was supported in part by funds for theresearch project conducted by the National Agriculture and Food Research Organization.
References[1] M. Oshige, S. Kawasaki, H. Takano, K. Yamaguchi,
H. Kurita, T. Mizuno, S. Matsuura, A. Mizuno and S.
Katsura, J. Fluorescence, 21, (2011) 1189
[2] H. Yasuda, T. Miura, H. Kurita, K. Takashima, and A.
Mizuno, Plasma Proc. Polymers, 7, (2010) 301[3] S.Ikawa, K.Kitano and S.Hamaguchi, Plasma Proc.
Polym., 7(1), (2010) 33