Low temperature air plasma sterilization on Pseudomonas
aeruginosa
Junling Gao, Jierong Chen, and Liqing Yang
School of Energy and Power Engineering, School of Energy and Power Engineering, School of Life Science
and Technology, Xi’an Jiao Tong University, Xi’an, 710049, China
Abstract: Low temperature air plasma was used to sterilize the Pseudomonas
aeruginosa (P. aeruginosa ATCC15442) samples on the polyethylene
terephthalate (PET) sheet in a self-designed reactor that included the
discharge area, afterglow area and remote area. The air plasma sterilization
rules in the three zones were respectively showed on the following conditions:
discharge power was 60W and gas flux was 30 cm 3 / min. For a treatment
time of 90 s, the germicidal effects (GE) were 4.01, 3.87 and 2.91. The
results showed that low temperature air plasma can kill effectively
P.aeruginosa in a short time and have different sterilization effects in
different reactor zones. Before and after plasma treatment, scanning
electron microscopy (SEM) was used to observe changes in cell morphology.
The cell walls or cell membrane cracking was testified by determining the
content of protein using coomassie light blue technique. The results show the
plasma activity cracking the cell wall and cell membrane, and resulting in
cellular content leakage. We suggest the cell membrane damage is
possibly the mechanism to kill P. aeruginosa under the plasma.
Keywords: Low temperature air plasma; Pseudomonas aeruginosa;
Germicidal effect; Mechanisms
1. Introduction
Low-temperature plasma technology meets the
demands for development of new technologies
having some advantages such as safety,
convenience, and lack of residual toxicity to
sterilize medical devices and instrumentation
including those viruses and pathogens such as
Pseudomonas aeruginosa. P. aeruginosa is mostly
a nosocomial opportunistic pathogen. And as the
increasingly multidrug resistant, the infection
incidents have occurred frequently in recent years
[1-2]. The primary methods now such as those
using autoclaves, ovens, chemicals like ethylene
oxide (EtO) and gamma irradiation process, are
associated with some level of damage to the
material supporting the microorganisms and have
raised public controversies concerning their
environmental effects and other health issues.
Plasma sterilization based on different technologies
eliminates those drawbacks, and also clearly shows
the effectiveness in killing bacteria [3-4].
In this paper, because of the special reactor
structure, the remote-plasma reactor consists of a
discharge area, an afterglow area and a remote area
in remote air plasma field. In the three areas, the
germicidal effects(GE)and inactivation mechanisms
of P. aeruginosa ATCC 15442 on the surfaces of
medical Polyethylene Terephthalate (PET) films
were reveaed.
2. Experimental
2.1. Gas plasma generator
A self-designed ideal tube-reactor was used (Figure
1). This reactor includes five parts: gas inlet,
reaction chamber, gas exhaust, power supply (SY500W 13.56 MHz) and matching network (SP-II
matcher); These are made by the Science Academy
of China. In the reaction chamber (Figure 2), a
GE  lg N 0  lg N t
these N 0 and N t
are the numbers of
colony-forming units of the control and sterilized
samples, respectively [5].
Figure 1. Schematic structure of low temperature plasma
reactor.
(1) Gas bottle; (2) valve; (3) mass flow meter; (4) inductance
coil; (5) reaction chamber; (6) sample; (7) vacuum gauge; (8)
electromagnetism valve; (9) vacuum pump; (10) RF generator;
(11) grounding protection; (12) matching system.
To learn the relative contribution of ultraviolet
radiation in air plasma sterilization process, some
samples were treated under the filter of lithium
fluoride (LiF) at the same above conditions. The
bottle is a special, which can effectively prevent
the interference of other reactive particles in the
plasma field and leaves only λ ≥ 120nm of
ultraviolet photons passing, so the bacteria in the
tube are effected only by the UV [6,7].
2.3. Cell Morphology changes observed by SEM
Figure 2. Schematic structure of three areas in the plasma
field.
Pyrex glass tube (length 1000 mm, diameter 45 mm)
where inductively coupled RF discharge is excited,
consists of a discharge area, an afterglow area and a
remote area.
2.2. Germicidal tests
A discharge area, an afterglow area and a remote
area in the reaction chamber were achieved in
remote air plasma field on the following conditions:
discharge power was 60 W and gas flux was 30
cm 3 /min. And the discharge area is the distance
within the 40 cm from the center of induction coil
and over 60 cm is a remote area, the middle one is
an afterglow area. Medical PET films which were
cut to the size of 50 mm × 25 mm and
contaminated by P. aeruginosa ATCC 15442 as the
samples were positioned on a carrier and directly
exposed to the plasma in three zones for 10-120 s.
After plasma treatment, the surviving bacilli were
removed from the samples into phosphate buffer
solution (PBS), they were transferred to Petri dish
which contained nutritional agar and incubated at
37.8 ℃ for 24 h prior. Count the final colonies
and calculate the Germicidal effect (GE), which
was determined by the following equation:
A scanning electron microscope (SEM, JSM–6700F,
Japan) was used to observe the morphology of the
bacteria before and after the plasma treatment. The
P. aeruginosa samples on the PET were fully
exposed to plasma and placed in different zones of
the air plasma field at certain distances of 15 cm
(discharge zone), 45 cm (afterglow zone), and 75
cm (remote zone) from the center of the induction
coil for a treatment time of 90 s .And then The
samples were shown at a magnification 10,000×
under scanning electron microscope.
2.4. Determination of protein leakage quantity
Coomassie light blue technique was used to test the
content of protein after air plasma treatment.
Samples with P. aeruginosa being treated directly
by air plasma were eluted in the PBS to form
suspensions of bacilli. These suspensions were
centrifuged, then put the upper pellucid solution to
mix with the coomassie brilliant blue dye and
placed for 3 min at room temperature. Absorbency
of this mixed solution was determined at the wave
length 595 nm by colorimetry. The quantity of
protein leakage of the cell after sterilization could
be gained by putting the determined absorbency
value in the regression equation of the standard
curve of bovine serum albumin [8,9].
3. Results and discussion
3.1. Germicidal effect
4.5
4.5
4.0
3.5
3.5
3.0
2.5
2.0
discharge area
afterglow area
remote area
1.5
1.0
0.5
Germicidal effect(GE)
Germicidal effect(GE)
4.0
3.0
2.5
Plasma treatment(discharge area)
Plasma treatment(afterglow area)
Plasma treatment(remote area)
Under filter of LiF(discharge area)
Under filter of LiF(afterglow area)
Under filter of LiF(remote area)
2.0
1.5
1.0
0.5
0
20
40
60
80
Plasma treatment time / s
100
120
Figure 3. Effect of plasma treatment time on germicidal
effect at three different areas in air plasma.
(Power: 60W; Air flux: 30 cm 3 /min;)
The sterilization effect of P. aeruginosa directly
exposed to air plasma is clearly shown in the Figure
3. It is well known that the germicidal effects (GE)
have a strong dependence on the plasma treatment
time and different areas. With the time lengthening,
germicidal effect values of P. aeruginosa increase.
In the discharge area the GE is higher than the ones
in the afterglow area and the remote area, and
increases rapidly with an increase of treatment time.
For a treatment time of 90s, the GE are respectively
4.01, 3.87 and 2.91 in the three areas. For 100-120
s, the GE values are stable as being close to
complete reaction. The obtained values indicate
that air plasma can effectively inactivate P.
aeruginosa within the discharge area and the
afterglow area within a short time.
Comparison of germicidal effect between the
ultraviolet radiation in the air plasma and the
integration of reaction particles is shown in Figure
4, from which, the contribution of UV is clearly
known. In the discharge area, the GE values of UV
are 0.41 ~ 0.72, accounting for 16.5% to 23.4% of
the total. The results indicate that UV adiation is
not a dominant sterilization agent in this process.
3.2. Sterilization mechanisms of air plasma
SEM was used to observe changes of P. aeruginosa
in cell morphology before and after plasma
treatment. SEM micrographs in Figure 5 were taken
before and after plasma treatment and the treatment
time was 90 s. Before plasma treatment (Figure
5-A), intact cells and complete cellularity can be
0.0
0
20
40
60
80
100
Plasma treatment time / s
120
Figure 4. Comparison of germicidal effect between ultraviolet
radiation in the air plasma and integration at distance of (a)
15 cm; (b) 45 cm; (c) 75 cm.
(Power: 60W; Air flux: 30cm 3 /min;)
Figure 5. Scanning electron micrographs of Pseudomonas
aeruginosa. (a) Before plasma treatment (b) discharge area
(15 cm) (c) afterglow area (45 cm) (d) remote area (75 cm)
(Power: 60 W; Treatment time: 90 s ; Air flux: 30 cm 3 /min;)
observed. In the discharge zone (Figure 5-B),
almost no cells remain intact, but a mass of debris
and leakage is observed. In the afterglow zone
(Figure 5-C) some cells crack and some are etched
and display small holes in the cell wall. In the
remote zone (Figure 5-D), some cells are deformed,
but most cells remain intact with no cell content
leakage. SEM provides the best proof of destruction
of the microorganism cell walls during the various
processes and cell wall or cell membrane cracking
is the reason resulting in P. aeruginosa death. They
also show that air free radicals play an important
role in cracking cell wall or cell membrane.
Figure 6 shows the quantities of protein leakage
after plasma treatment. The results exhibit the
-1
Protein leakage quantity /mg. mL
0.266
0.264
0.262
0.260
0.258
0.256
0.254
0.252
0.250
0.248
0.246
0.244
0.242
0.240
receives extensive attention and is considered as a
promising sterilization technology.
Acknowledgements
discharge area
afterglow area
remote area
10
15
30
60
90
Plasma treatment time /s
120
Figure 6. Quantities of protein leakage at the different zones
after sterilization.
(Power: 60 W; Air flux: 30 cm 3 /min;)
different degrees leakage in different zones, and
further prove that the plasma induces cell wall or
cell membrane some degree of cracking, resulting
in changes in cell permeability, content substances
leaking in the cell. The leakage is maximum in the
discharge zone.
This work was supported by the National Natural
and Science Foundation of China (Nos. 30571636
and 20877062), by the Specialized Research Fund
for the Doctoral Program of Higher Education (No.
20060698002), by the key Scientific Technique
Special item of ‘‘13115’’ Innovation Project in
Shaanxi Province 2008ZDKG-78 and the key
Scientific Technique item of Shuzhou City SG0842.
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4. Conclusions
This study make a preliminary discussion for air
plamsa killing Pseudomonas aeruginosa on the
PET under the following conditions: discharge
power was 60 W and gas flux was 30 cm 3 /min, the
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than 120 seconds through low-temperature air
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