22P078-P
Effects of VUV/UV Emission and Radicals on Inactivation of Spore Forming
Bacteria by Surface-wave Excited Nitrogen/Oxygen Plasma
表面波励起窒素・酸素プラズマを用いた芽胞形成菌不活化におけるVUV/UV放射およびラジカルの効果
Akira Tsukasaki, *Ying Zhao, *Akihisa Ogino and *Masaaki Nagatsu
塚崎 旭, *趙 穎，*荻野 明久，*永津 雅章
Graduate School of Engineering, Shizuoka University, 3-5-1, Johoku, Hamamatsu 432-8011, Japan
静岡大学大学院工学研究科電気電子工学専攻，〒432-8011 浜松市城北3-5-1*Graduate School of Science and Technology, Shizuoka University, 3-5-1, Johoku, Hamamatsu 432-801 1, Japan*静岡大学創造科学技術大学院，〒432-8011 浜松市城北3-5-1
In general, under plasma exposure, bacterial cells can be destroyed by synergistic reaction of UV radiation,
plasma particles and so on. Insight into each factor how to effect on bacterial and the processing are thegoal of this study. In this work, we focus on the role of radicals and VUV/UV emission as a sterilization
agent of Geobacillus stearothermophilus spores. The measurements of colony counting method andscanning electron microscope were used to investigate the effect of radicals and VUV/UV emission indetail.

1. Introduction
Currently, many sterilization techniques, forexample autoclave, H 2O2 gas sterilization andradiation sterilization are used in medicalfacilities. However these techniques haveproblems, such as high temperature, toxicity andhigh cost, respectively and proper use is carriedout according to the purpose. And plasma
sterilization mechanisms are studied to establish
the low temperature plasma as a more effectivesterilization tool.
Boucher et.al reported that UV photons in theplasma have an important role[1]. And
Bol’shakov et.al reported that UV photons andoxygen radicals synergistically induce thechemical decomposition of microbial cell. Moisanet.al suggested that chemical reactions, UV
emission, photodissociation and ion sputtering aresynergistically effect, and there is the inactivation
effect of spores clearly higher than the case where
each acts alone[3]. And the extent of theinfluence of each factor depends on the plasma
operating parameters such as power and gas
mixture and flow rate. In our previous work, a
six-log reduction in spores could be achieved
onlyseveralminutes
irradiation
withlow-pressure oxygen/air simulated surface-wave
plasmas and the chemical etching reaction fromthe reactive oxygen radicals make more efficient
inactivation rate, and in oxygen/nitrogen mixture
plasma, the best efficiency of etching wasappeared in the case of 90% oxygen mixture with
10% nitrogen[4-6]. But we don’t know how

VUV/UV damages the DNA in the spore andwhat relation between nitrogen radicals and theinactivation of spores in our previous works. In
this work, we aimed to investigate effects ofVUV/UV emission to DNA and nitrogen radicals
on inactivation of spore forming bacteria.
2. Experimental setup
The experimental setup used for the sterilization
tests consists of a stainless steel cylindrical vacuum
chamber having a diameter of 300 mm and a heightof 300 mm with a microwave launcher and 2.45
GHz microwave generator, as shown in Fig. 1. The
plasma is produced at a pressure of 13 Pa and a
total gas flow of 100 sccm by a microwave powersource. The launched microwave power varied from
200 to 800 W. The optical emission spectroscopy
was used to diagnose the plasma parameters. We
used the biological indicator (BI) of G.
stearothermophilus spores with a population of
2.1×106 pasted on a small stainless disc, which was
put on the sample stage and treated with oxygen
plasma or nitrogen plasma. The sample stage wasabout 10 cm below the quartz window. After
treatment, one set of treated samples were washedwith 2 ml of BHI (Brain-Heart Infusion) solution in
the test tube for colony counts. Test tubes
containing the biological indicator carriers werestirred for 1 min at room temperature. After anappropriate dilution, 0.1 ml of the spore suspension
was inoculated onto nutrient agar media and after
two days of incubation at 55 ºC, the colony forming
units (CFUs) corresponding to each surviving cell,were counted. The CFUs reduction of treatedsamples were compared with untreated sample andplotted as the survival curve. In parallel, a scanningelectron microscope was used to observe themorphologies of the bacteria before and after theplasma treatment.
Fan
2.45GHz
Slot antenna

400W

ExposureWith filter

600W

ExposureWith filter

800W

l ExposureWith filter

microwaveplasma
LiF filterz

BiologicalIndicator

Metal caseFig.2 Survival curves of spores treated by O2
plasma without and with a LiF filter

Fig.1 Schematic view of experimental setup of
the surface-wave plasma device.

4. Conclusion
In this research, we studied the relation between
the effects of radicals and VUV/UV radiation andthe inactivation of spores in surface-wave plasma.
The other results will be shown in the furtherreport.

106■ 400W▲ 600W● 800W

Exposure

105

CFUs

104
103
102
101
100
10-1
0

30

60

90

120

150

180

210

240

270300Treatment Time [s]107106□ 400W△ 600W○ 800W

With LiF filter

105
104

CFUs

3. Results and Discussion
Figure 2 is the result of CFUs of Geobacillus
stearothermophilus spores treated by oxygen
plasma without and with LiF filter to block particles,
such as radicals, at 400 W, 600 W, and 800 W,respectively. When the LiF filter was set above theB.I. samples, the inactivation efficiency wasobviously decrease. This result might suggest thatthe main inactivation factor is oxygen radicals inoxygen plasma.
Figure 3(top) is the survival curves of spores
treated by the direct irradiation of nitrogen plasma
at 400 W, 600 W, and 800 W, respectively. AndFig.3(bottom) is the result of the survival curvewhen the plasma particles is intercepted by the LiF
filter which cutoff wavelength is 120 nm in thesame plasma condition. The differences in survival
curves obtained at each condition were less thanthat in the case of oxygen plasma. That resultmeans the dominate factor of the deactivation isclearly different from oxygen plasma treatment. In
the case of nitrogen plasma, the dependence ofplasma particles on the deactivation is much less.
The main sterilization mechanism is VUV/UVradiation. However, when the microwave power
was 800 W, six-log reduction time shown in Fig.
3(top) was shorter than that at 400 or 600 W. We
will investigate the effect on spores only by radicals(neutrals) and VUV/UV radiation without any
charged particles by inserting the grounded porous
metal electrode between plasma and sample
surfaces. And it will become possible to investigate
the effect of radicals by the existence of a LiF filter.

107

103
102
101
100
10-1
030

60

90

120

150

180210240270300Treatment Time [s]Fig.3 Survival curves of spores treated by N2plasma without (top) and with (bottom) a LiF filter

Acknowledgments
This work was partly supported by Grant-in-Aid
for Scientific Research from Japan Society for thePromotion of Science (JSPS).
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