Treatment of enterococcus faecalis bacteria by a helium atmospheric cold plasma brush with oxygen addtion

Treatment of enterococcus faecalis bacteria by a helium atmospheric cold
plasma brush with oxygen addition
Wei Chen, Jun Huang, Ning Du, Xiao-Di Liu, Xing-Quan Wang et al.

Citation: J. Appl. Phys. 112, 013304 (2012); doi: 10.1063/1.4732135
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Published by the American Institute of Physics.

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Downloaded 09 Aug 2012 to 159.226.35.234. Redistribution subject to AIP license or copyright; see />Treatment of enterococcus faecalis bacteria by a helium atmospheric
cold plasma brush with oxygen addition
Wei Chen,

1,a),b)
Jun Huang,
1,a)
Ning Du,
2
Xiao-Di Liu,
2
Xing-Quan Wang,
1
Guo-Hua Lv,
1
Guo-Ping Zhang,
1
Li-Hong Guo,
2
and Si-Ze Yang
1,3
1
Key Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics,
Institute of Physics, Chinese Academy of Science, 100190 Beijing, China
2
Department of Oral Biology, Peking University School and Hospital of Stomatology, 100080 Beijing, China
3
Fujian Key Laboratory for Plasma and Magnetic Resonance, Department of Aeronautics, School of Physics
and Mechanical and Electrical Engineering, Xiamen University, Xiamen 361005, China
(Received 3 April 2012; accepted 30 May 2012; published online 6 July 2012)
An atmospheric cold plasma brush suitable for large area and low-temperature plasma-based
sterilization is designed. Results demonstrate that the He/O
2
plasma more effectively kills

Enterococcus faecalis than the pure He plasma. In addition, the sterilization efficiency values of
the He/O
2
plasma depend on the oxygen fraction in Helium gas. The atmospheric cold plasma
brush usin g a proper ratio of He/O
2
(2.5%) reaches the optimum sterilization efficiency. After
plasma treatment, the cell structure and morphology changes can be observed by the scanning
electron microscopy. Optical emission measurements indicate that reactive species such as O
and OH play a significant role in the sterilization process.
V
C
2012 American Institute of Physics.
[ />I. INTRODUCTION
In recent years, nonthermal atmospheric pressure plas-
mas have been widely studied for several novel applications
in biomedicine
1–16
and nanotechnology.
17,18
Among the
novel applications, sterilization by low temperature atmos-
pheric pressure plasmas, which are partially ionized gases, is
attracting significant attention.
19–22
Very recently, a room-
temperature, battery-operated, handheld air plasma jet was
designed and applied to effectively inactivate multilayered
Enterococcus faecalis biofilms by Lu and co-workers.
23

Enterococcus faecalis is a Gram-positive facultative anaero-
bic bacterium, which has a strong adaptability to temperature
and resistance to multiple antibiotics. The conventional steri-
lization methods such as heat and chemical agents are not
suitable to treat these notorious heat- and drug-resistant
pathogens. By contrast, low temperature atmospheric pres-
sure plasma is a more effective technology, which can pro-
vide many chemically and biologically active species,
including various atoms, ions, energetic electrons, metasta-
ble particles, and UV irradiation. It has been shown that
when these plasmas generated at atmospheric pressure are
used for sterilization, they do not cause bulk destruction of
living tissue, do not damage heat-sensitive materials, and
may be touched by humans without any harm.
1
However, the
available atmospheric plasma sterilization processes in cur-
rent medical uses have some drawbacks that the treatment
sizes from plasma jets,
24
plumes,
25
or plasma needles
26
with
a diameter of a few millimeters or less are rather small.
In this paper, we report an experimental study on inacti-
vation of Enterococcus faecalis bacteria by means of an
atmospheric cold plasma brush driven by an ac power
supplier. The influences of oxygen addition into the helium

atmospheric plasma brush on its sterilization capability are
studied. After the plasma treatment, scanning electron mi-
croscopy (SEM) is used to inspect the bacterial cell structure
changes. According to the optical emission spectra of the he-
lium atmospheric plasma brush with and without oxygen
addition, the roles of the various plasma agents in the inacti-
vation of bacteria are investigated in detail.
II. EXPERIMENT
A. Atmospheric cold plasma brush
Figure 1(a) is a schematic diagram of the experimental
setup. Our atmospheric cold plasma brush comprises of two
parts: a discharge chamber and two electrodes placed outside
the discharge chamber. The discharge chamber with a vol-
ume of 80 Â 35 Â 1mm
3
is made of quartz. Both electrodes
are made of copper foil of 10 mm wide wrapping the dis-
charge chamber. The gap between the inner edges of the cop-
per foil is 15 mm. The ground electrode is on the upstream
side; the active electrode is on the downstream side and
10 mm apart from the chamber nozzle. A sinusoidal ac high-
voltage (11 kHz, 22 kV peak to peak) is applied between two
electrodes for the exci tation and sustaining of the discharges.
High-purity helium and oxygen are used as the working gas,
and the flow rate is controlled by the flow meter. Throughout
the experimental procedure, the applied power (P) is fixed at
24 W, and the He flow rate always maintains 4500 SCCM
(SCCM denotes standard cubic centimeter per minute). The
separation between the nozzle and the sample is 5 mm. Fig-
ure 1(b) shows the discharge photograph of the atmospheric

cold plasma brush with 4500 SCCM helium (P ¼ 24 W). The
visually uniform brush-shaped plasma is formed and
extended out of the discharge chamber through the narrow
slit at outlet.
a)
Wei Chen and Jun Huang contributed equally to this work.
b)
Author to whom correspondence should be addressed. Electronic mail:

0021-8979/2012/112(1)/013304/4/$30.00
V
C
2012 American Institute of Physics112, 013304-1
JOURNAL OF APPLIED PHYSICS 112, 013304 (2012)
Downloaded 09 Aug 2012 to 159.226.35.234. Redistribution subject to AIP license or copyright; see />B. Sample preparation
An overnight culture containing approximately 10
7
CFUs/ml is prepared (CFUs denotes colony-forming units).
Then, with filter papers as the supporting media, 5 lL of sus-
pension containing Enterococcus faecalis bacteria are
dropped onto sterilized filter papers (5 mm diameter), and
the papers are allowed to dry in a moderate vacuum incuba-
tor at 37

C for 1 h. Afterward, the filter papers containing
Enterococcus faecalis bacteria are exposed to the atmos-
pheric cold plasma brush sustained with He/O
2
gas. After
plasma treatment, the plasma treated filter papers are trans-

ferred into a 1.5 ml centrifuge tube contain ing 1 ml physio-
logical saline and mixed using a vortex mixer for 1 min. The
bacterial strains are grown into the sterile Brian Heart Infu-
sion agar, and the number of the living bacteria cells is
counted after incubation at 37

C for 48 h.
III. RESULTS AND DISCUSSION
A. I–V curve
Voltage is measured using a 1000:1 high voltage probe
(Tektronix P6015A, maximum input voltage: DC 20 kV,
bandwidth: 75 MHz). For current measurements, a magnetic
core current probe (Tektronix P6021, maximum discharge
current: 15 A, bandwidth: 60 MHz) is utilized. Signals from
the current and voltage probes are acquired and recorded by
a digital Tektronix TDS 210 oscilloscope. It can be seen
from Fig. 2 that the breakdown of working gas in atmos-
pheric cold plasma brush, resulting in a large number of cur-
rent filaments, the so-called micro-discharges, which are
randomly distributed both in time and space. In this filamen-
tary mode,
27–29
the discharge starts with local gas breakdown
at many points in the discharge volume. This mode is charac-
terized by a periodic current constituted by many discharge
pulses in each half cycle. An inverse current peak is also
observed when the polarity of the applied voltage changes.
B. Sterilization effect of atmospheric cold plasma
brush
Figure 3 shows the change of the survival curves of

Enterococcus faecalis bacteria in the He with addition of dif-
ferent amounts of O
2
(1%, 2.5%, 5%, 10%) into atmospheric
cold plasma brush (P ¼ 24 W). As can be seen in Fig. 3,a5
log reduction of the cells required only 30 s exposure time
with about 2.5% oxygen addition into the helium plasma,
while 90 s were required in the pure helium plasma. It was
also noted that 2.5% O
2
addition provided the faster killing
speed for a complete kill of the bacteria within 60 s exposure
time, while it needed 120 s to completely kill the bacteria
using pure helium plasma, as shown in Fig. 3. The experi-
mental data shows that sterilization efficacy of He/O
2
plasma
is better than that of pure He plasma.
C. Scanning electron microscopy
Scanning electron microscopy (SEM) (Model S-5200,
Hitachi, Japan) was used to examine the morphology and
structural changes of Enterococcus faecalis after plasma ex-
posure. The controlled and the plasma treated samples were
placed in the fixation and then coated with a thin layer of
plasma sputtered platinum. Figure 4 shows the SEM
FIG. 2. Typical oscillograms of applied voltage and discharge current of a
filamentary discharge in He/O
2
FIG. 3. Survival curves of Enterococcus faecalis bacteria in the He with dif-
ferent O

2
additions into atmospheric cold plasma brush (P ¼ 24 W).
FIG. 1. (a) Schematic diagram of the experimental setup. (b) Photograph of
the atmospheric cold plasma brush with 4500 SCCM He (P ¼ 24 W).
013304-2 Chen et al. J. Appl. Phys. 112, 013304 (2012)
Downloaded 09 Aug 2012 to 159.226.35.234. Redistribution subject to AIP license or copyright; see />photographs of untreated control and plasma treated Entero-
coccus faecalis with pure helium plasma brush and with oxy-
gen addition at an amount of 1% and 2.5%, respectively. It
can be seen from Fig. 4 that the plasma treatment of Entero-
coccus faecalis resulted in a significant alteration in cell
structure and morphologies when compared with the
untreated controls. As seen from images in Figs. 4(b)–4(d),
more structure damages were observed on Enterococcus fae-
calis with increasing the oxygen addition amount with vol-
ume percent of 0, 1%, and 2.5%. Consistent with the cell
surviving curve shown in Fig. 3, He plasma with 2.5% O
2
addition provided a much faster killing of the bacteria. From
Fig. 4, it was also noted that the oxygen-based plasma spe-
cies were mainly responsible in improving the plasma inacti-
vation efficiency and oxidation capacity of Enterococcus
faecalis.
D. Optical emission spectra
To identify the various reactive species generated by the
atmospheric cold plasma brush, the optical emission spec-
trum (Stellarnet, EPP-2000 C) of He/O
2
plasma is measured
in the 200–850 nm wavelength range at atmospheric pres-
sure. Figure 5 shows the optical emission spectra of the

plasma with 4500 SCCM He/O
2
(2.5%) taken at 5 mm bot-
tom the nozzle along axis. It is well known that UV radiation
in the 200–300 nm region with doses of several milliwatt per
square centimeter may cause lethal damage to cells. How-
ever, the UV radiation intensity in 200–300 nm wavelength
range is below 50 lW/cm
2
. In addition, the inactivation
effect on bact eria by ultraviolet radiation is mostly related to
the DNA/RNA damage in UV-C (200–280 nm).
30
Therefore,
the UV emission plays a minor role in the inactivation of
the bacteria. It clearly shows that excited O lines (285 and
777 nm), OH line (309 nm), molecular nitrogen lines
(C
3
G
u
—B
3
G
g
) (316, 337, 357, 380, and 391 nm), O
þ
line
(427 nm), H
a

line (656 nm) and excited He atom lines (501,
587, 640, 669, 707, and 729 nm) are presented in the
plasma brush. Atomic oxygen is probably due to the direct
electron impact dissociations of oxygen molecule (e
À
þ O
2
! O þ O þ e
À
).
31
O may also be formed through Penning
ionization (N
2
þ O
2
! N
2
þ O þ O).
32,33
We assign the fea-
ture at 309 nm to the OH (A
2
R
þ
—X
2
G) transition;
34,35
the

formation of which is attributed to the reaction of excited O
with water vapor (H
2
O þ O
*
! 2OH) and the electron
impact dissociation (H
2
O þ e
À
! H þ OH þ e
À
). The pres-
ence of N
2
(C!B) lines clearly reveals the air entrainment
in the plasma brush. The H
a
emission line at 656 nm is
formed by the collision between water vapor molecules
and electrons (H
2
O þ e
À
! H þ OH þ e
À
). The formation of
O
2
þ

is probably due to Penning ionization (He
*
þ O
2
! He þ O
2
þ
þ e
À
).
36
Furthermore, the addition of oxygen to
the He plasma decreases He molecules’ emission,
37
but
enhances intensity level of reactive oxygen radicals. This
can be accounted for from that some electrons are probably
consumed to produce O radicals. Others then collided with
air, water vapor, and He to produce N, OH and He radicals.
In fact, reactive oxygen species (ROS) play a major role in
FIG. 4. Scanning electron micrographs of Enterococ-
cus faecalis bacteria of (a) untreated control, (b) helium
plasma treatment, (c) He þ 1% O
2
plasma treatment,
(d) He þ 2.5% O
2
plasma treatment. Plasma conditions
were 4500 SCCM helium flow rate and 60 s exposure
time (P ¼ 24 W).

FIG. 5. Emission spectra of the plasma with 4500 SCCM He/O
2
(2.5%)
taken at 5 mm bottom the nozzle (P ¼ 24 W).
013304-3 Chen et al. J. Appl. Phys. 112, 013304 (2012)
Downloaded 09 Aug 2012 to 159.226.35.234. Redistribution subject to AIP license or copyright; see />bacterial inactivation
38,39
and lead to various biological
effects in the intracellular space.
40
These reactive species
could directly act on microorganisms, especially their outer
membranes, and damage them because of a strong oxidation
mechanism. To increase the content of OH and O radicals,
increasing O
2
concentration is a useful method.
IV. CONCLUSION
In this study, an atmospheric cold plasma brush is
designed and fabricated. The effect of oxygen on the deacti-
vation of Enterococcus faecalis by an atmospheric cold
plasma brush is presented. Experimental results show that
oxygen addition into the plasma brush can improve the deac-
tivation efficiency of Enterococcus Faecalis. The atmos-
pheric cold plasma brush using a proper ratio of He/O
2
(2.5%) reaches the maximum sterilization efficiency. After
plasma treatment, SEM images show obvious cell structure
damages in morphology of the organisms. In addition, opti-
cal emission spectroscopy clearly indicates that there are

excited OH, O, N
2
, and He in the atmospheric cold plasma
brush, and OH and O radicals are mainly responsible for the
bacteria death.
ACKNOWLEDGMENTS
This work was supported by the Young Scientists Fund
of the National Natural Science Foundation of China under
Grant No. 11005151.
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