22P074-P
Bacteria Sterilization by High Electric-Field Plasma without Discharges for MedicalApplications
医療応用としての高電場プラズマによる殺菌
Fang-Yu Fana,b,c, Chung-Ming Liua,d,e, Chao-Chia Wengf, Yasushi Nishidaa,g, andKeng-Liang Oua,f,haResearch Center for Biomedical Devices, Taipei Medical University, Taipei 110, Taiwan
Graduated Institute of Dental Technology, Taipei Medical University, Taipei 110, Taiwan
cDepartment of Mechanical Engineering National Taiwan University
dDepartment of Chemical and Material Engineering, LungHwa University of Science and Technology, Taoyuan 306,TaiwaneGraduate School of Engineering Technology, LungHwa University of Science and Technology, Taoyuan 306,
Taiwanf
Graduated Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei 110,
TaiwangPlasma and Space Science Center, National Cheng Kung University, Tainan 70101, Taiwan
hResearch Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110,
Taiwanb

A comprehensive research for sterilizing bacteria by means of high electric-field plasma without apparent
discharges between electrodes is carried out. The most common pathogens responsible forhospital-acquired infections are Staphylococcus aureus and Escherichia coli. The experimental results
showed that these bacteria were sterilized up to 99 % S. aureus and 83 % E. coli within 60 second. The
high electric-field plasma system can induce structural damage and ruptures on bacterial cells leading to
the death of the bacteria.

1. Introduction
Using an effective sterilization method to control
environment infections of human life has received
muchattention.
Traditionalsterilization
technologies are the application of heat, chemicalmaterials and/or irradiation to UV light or X-rays.
Although these methods have been widely used inindustries, healthcare and medical fields, but eachmethod has its application limit. A highelectric-field plasma (HEFP) technique in an
atmospheric pressure has been developed to control
and keep the environmental atmosphere in clean
state, such as sterilization [1]. In this study, we will
present the results of high electric-field plasma
treatments of two types of bacteria. The sharp
rise-time strong electric field formed in between themetal-plate electrodes shows no apparent
discharges, but a few amount of electron currents
are flowing. This system is free from the effects byozone, NOx, OH*(OH radical) or UV lightemission. Those toxic materials do affect strongly
to all of any materials or circumferences including
human body.
2. Materials and Methods
In this study, the applied maximum voltage is 5.0
kV and the peak current is about 0.5-1.0 mA in theHEFP system. The antibacterial procedure wasaccording to JIS Z2801:2000 standard [2-3]. Twotypical bacteria used as test strain for thisexperimental study are the Gram-positive
Staphylococcus aureus (ATCC6538P: S. aureus)
and Gram-negative Escherichia coli (BCRC11634:E. coli). These microbes are commonly used as
research sites for environmental indicators.
A single colony was incubated for 18 h to 24 h
at 37oC on nutrient agar plates (Difco, USA). The
bacterial cells were standardized to concentration of
2.5 ×105~1.0×106 CFU/ml under sterile conditions
by optical spectrophotometry (Thermo, USA).
Portions of 10 μl of the standardized concentrationsuspensions were evenly spread on the dielectricglass substrates. The each sample are
placing/inserting at the center of the metal-plateelectrodes with 1.2 mm gap spaces. The samples
were treated by HEFP with four different treatment
time of 0, 15, 30, 45 and 60 second, in which eachcondition had replication for three times. The
number of colonies was calculated to theantibacterial rate (AR). To investigate the effects ofdamages caused by HEFP treatment on bacteria
cells, the Scanning electron micrographs (SEM)
was used to inspect morphologies of S. aureus andE. coli cells.
3. Results and discussion

The SEM images of S. aureus is shown in Fig. 1
at each time duration after HEFP treatment. Theseimages indicate that the outer membrane rupturesand splits matrix and distortion of the dead cells
after plasma treatment. The SEM observation on E.
coli in Fig. 2 also shows similar results. The HEFP
treatment is verified, showing very effective forbacteria sterilization.
(a)different timing on HEFP sterilization. Therefore,
the percentage for an effective sterilization could beachieved at 30 second of treatment. However, S.
aureus shows better sterilization than E. coli, which
could be seen clearly after 60-second treatment, i.e.
99 % of initial S. aureus is effected by HEFP
treatment. It was noted that the response of S.
aureus was significant than E. coli.

(b)

Fig.1. The results of Scanning electron micrographs of S.
aureus at different time duration in HEFP treatment. (a) 0s (b) 60 s.
(a)

(b)

Fig.2. The results of Scanning electron micrographs of E.
coli at different time duration in HEFP treatment. (a) 0 s(b) 60 s.

The bacterial of S. aureus (gram positive) has 3layers as cell membrane, including plasmamembrane, periplasmic space and peptidoglycan
from inside of the cell. In the case of E. coli,
however, it has an outer membrane which acts as aprotective layer for the Gram-negative microbes[4-5]. Therefore, it may be rather easier to destroy
the cell membrane in case of S. aureus. However, it
is of great importance for practical systems for theanalysis that the antibacterial rate (AR) could bedetermined by Eq. (1) as recognized for
antibacterial effect.
N N treated.
(1)
AR (%)  100  control
N control
Here, Ncontrol is number of bacteria adhered on
untreated samples after 24 h incubation, and Ntreatedis number of bacteria adhered on treated samples
after 24 h incubation.
The survival rates of these bacteria after
undergoing different treatment times are shown in
Fig.3. Results clearly show that continuous increaseof exposure time to HEFP perfectly led to thedecrease of survival rate of these microbes. The
antibacterial rate (AR) could be simplified for

Fig.3. Antibacterial rate of S. aureus and E. coli after
different treatment time.

4. Conclusions
This experimental analysis has been carried out
as part of a larger effort aimed for determining andunderstanding the fundamental processes involvedin new technique of HEFP sterilization. From ourresults on the experiments, effective measures on
the sterilization technique were clarified for both
Gram-positive and Gram-negative bacteria.
Acknowledgement
The authors appreciate to the Center ofExcellence for Clinical Trial and Research in
Neurology and Neurosurgery, Taipei Medical
University-Wan Fang Hospital, for their financial
support under the contract No. DOH100TD-N111-004.
References
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Trans. Plasma Sci. 39(2), (2011)717-24.
2. JISZ2801:2000, Japanese Industrial Standard (2001).
3. K.H. Liao, K.L. Ou, H.C. Cheng, C.T. Lin, and P.W.
Peng, Appl. Surf. Sci. 256, (2010)3642.
4. Block SS. "Sterilization" in Encyclopedia ofMicrobiology. New York, Academic, 4 (1992) 87 –103.
5. N. Papo and Y. Shai, J. Biol. Chem. 005 Mar 18; 280(2011) 10378-87.