Bacterial Decontamination on Surfaces
by Reduced-pressure Afterglow Plasma
Hongxia Liu and Yun Liu
Department of Environmental Science & Engineering, Xi’an Jiaotong University, Xi'an, P. R. China
Abstract: Afterglow argon plasma generated by RF discharge with 13.56-MHz
was used to sterilize the bacteria at reduced-pressure. Typical bacteria, grampositive Staphylococcus aureus (S. aureus) ATCC 6538, were used as test strains.
Bacteria cells held by cover-glass were placed at the different distance from the
plasma active discharge zone. By changing the RF power and exposure time, the
effects of argon plasma on bacterial inactivation were investigated. The
distribution of reactive species in the afterglow argon plasma were measured for
analyzing the individual action of electrons, ions and free radicals during
sterilization. Experimental results showed that S. aureus strains were killed
effectively within the distance of 0-30 cm under the conditions of plasma RF
power 100 W, exposure time 240 s and argon flow 20 cm3/min. It is concluded
that the charged particles in plasma play a dominant role during the inactivation
process because they can erode cell material, rupture cell membrane and then the
content of cells effuse. The germicidal kinetics of S. aureus by the afterglow
argon plasma was discussed to elucidatec the kill rates at different distance from
the active discharge zone.
Keywords: afterglow argon plasma; Staphylococcus aureus; sterilization
1. Introduction
Establishment of a sterilization and disinfection
technology that is safe and easy to apply is very
important in terms of prevention of infection,
especially for the aged or very young, who are less
resistant to infection. Plasma-based sterilization
techniques do not suffer from the problems for the
traditional techniques. Adequate processes are
efficient, do affect only slightly the bulk material,
are environmentally sound, do not produce toxic byproducts, are fast and cost effective [1,2].
In this study, we have introduced an afterglow argon
plasma generated by RF discharge with 13.56 MHz
for bacteria killing at reduced-pressure. Typical
bacteria, gram-positive Staphylococcus aureus (S.
aureus) ATCC 6538, were used as test strains. The
influence of the afterglow plasma treatment
conditions on the germicidal efficiency were
discussed. For analysing the individual action of
reactive species during sterilization clearly, double
Langmuir electron probe and electron spin resonance
(ESR) were used to verify the distribution of
electrons, ions and free radicals in afterglow argon
plasma. Finally, the germicidal kinetics of S. aureus
by the afterglow argon plasma was discussed to
elucidatec the kill rates at different distance from the
active discharge zone.
2. Experiments
2.1 Plasma Reactor
The experimental arrangement used is shown
schematically in Fig. 1. The reactor consisted of gas
inlet, reaction chamber, gas exhaust, power supply
(SY-500W 13.56 MHz) and matching network (SPⅡmatcher). The reaction chamber is a cylindrical
Pyrex glass tube (45 mm diameter, 1000 mm long),
where inductively coupled RF discharge is initiated.
Argon, with a purity of more than 99.99%, was used
as the work gas.
2.2 Argon Plasma Diagnosis
The double Langmuir electron probe was applied to
perform the argon plasma diagnosis. The leader of
this electron probe was connected with the plasma
reactor, and its location in reactor was regulable. The
measured V-A characteristics of the electrical probe
were used to determine the concentrations of
electrons and ions [3].
agar plates were then separately analyzed for colony
counts. Germicidal effect (GE) was determined by
the following equation [4],
In this experiment, clean and inartificial wool was
placed on different distance from the center of
induction coil to be treated and conserved for 24 h
at room temperature. Then ESR spectrometer (made
in BRUKER company, ESP-500) was used to
measure the concentration of free radicals in these
wool. The measurement conditions adopted were the
following: room temperature, microwave frequency
9.8 GHz, microwave power 3.177 mW, modulation
amplitude 0.2 mT, modulation frequency 100 kHz,
time constant 163.84 ms, scan time 163.84 ms.
Where N0 and Nt are the number of colony forming
units of control and exposed, respectively.
GE  log N 0  log Nt
(1)
3. Results and discussion
3.1 Distribution of Reactive Species in the
Afterglow Argon Plasma
Fig. 2 shows the distribution of reactive species in
the afterglow argon plasma. It indicates that the
concentration of free radicals changes little within
40 cm, whereas the concentration of electrons and
ions decline rapidly with increasing the distance,
they approximate to 0 at 30 cm. This happens
because different life-spans of various reactive
species [5]. This result indicates that electrons, ions
and free radicals can be separated at certain plasma
field, and then the roles of reactive species can be
analysed clearly during the afterglow argon plasma
sterilization.
Figure 1. Schematic view of experimental arrangement. (1) Gas
bottle; (2) Valve; (3) Mass flowmeter; (4) Inductance coil; (5)
Matching system; (6) RF generator; (7) Sample; (8) Reaction
chamber; (9)Vacuum gauge; (10) Electromagnetism valve; (11)
Vacuum pump; (12) Grounding protection.
2.3 Bacteria and Processing
The inactivation effect of the RF plasma was tested
on the S. aureus ATCC 6538. Colonies from
slanting nutritional agar (after 24 h of growth at
37°C) were picked up with a loop and diluted with
0.9% sterile saline to a turbidity equivalent to that of
0.5 McFarland standard (~108 CFU/mL). 0.01 mL of
this suspension was inoculated onto each 15 mm
square cover glass which was sterilized previously
and dried naturally in sterile Petri dishes at room
temperature. One of the cover glasses inoculated
with bacteria was kept as control (reference) which
was not exposed to plasma.
After plasma treatment, cover glasses were exposed
and control ones were put in 5 mL of phosphate
buffered saline (PBS) and oscillated. After
appropriately diluted of the formed eluent, 50 μL of
each dilution was inoculated onto nutritional agar
plates. After 48 h’s incubation at 35°C, nutritional
Figure 2. Relative concentration of electrons, ions and free
radical as a function of distance from the active discharge zone
(power: 90 W; exposure time: 3 min; argon flow: 20 cm3/min).
3.2 Germicidal Effect at Different Plasma
Treatment Conditions
In this study, we fixed on the argon flow as 20
cm3/min. The surface sterilization by the argon
plasma was investigated from the GE as a function
of the plasma RF power and the exposure time at a
constant distance of 0, 30 and 60 cm from the center
of the active discharge zone in the the reactor, as
shown in Figs. 3-4. Fig. 5. shows the typical effect
of the distance on the GE.
From Fig. 4, the GE enhance rapidly with increasing
the plasma exposure time in the whole argon plasma
reactor. Previous works [7,8] deemed that longer
argon plasma exposure time with higher RF power
can cause heavy degradation and damage to the
substrate surface. Therefore we find that the
optimum plasma sterilization conditions are 100 W
power and 240 s exposure time to obtain the
maximum GE of S. aureus on surfaces.
Figure 3. Effect of power on germicidal efficiency (exposure
time: 120s; argon flow: 20cm3/min).
In Fig. 3, the GE enhance with increasing the RF
power at the distance of 0 and 30 cm. The main
reason is that the ionization degree of argon gas and
average energy of reactive species are augmented
rapidly with increasing RF power. A mixed
atmosphere constituted by all reactive species
presents within 30 cm, especially the being of
charged particles, as shown in Fig. 2, make the
probability of action on cocci to be enhanced. The
GE at 60 cm is influenced little by RF power for
only free radicals of low concentration presented
here. Moreover, regardless of the RF power, the
distance is further, the GE value is lower. Compared
these conclusions with the distribution of reactive
species in Fig. 2, it can be concluded that the intense
etching action on cell of S. aureus by charged
particles, play a dominant role in the inactivation
process but not the free radicals. This etching action
erodes cell material, e.g. crust lipoprotein and inner
fat amylase of cell membrane, as a result, cell
membrane ruptures and content effuses, and causes
death of cocci finally [6].
Figure 5. Effect of distance on germicidal efficiency (power:
100 W; exposure time: 240 s; argon flow: 20 cm3/min).
Fig. 5 shows the GE as a function of the distance
from plasma active discharge zone under the
optimum sterilization conditions. The values in this
plot mean that argon plasma can inactivate S. aureus
effectively within 0-30 cm. The concentration of
electrons and ions is close to zero at about 30 cm, so
the etching action on substrate surface is inhibited
and reaction of free radicals is enhanced. Thus,
argon plasma sterilization in afterglow zone can
improve surface hydrophilicity, reduce surface
degradation and damage of substrate, especially for
medical materials [9,10].
3.3 Germicidal kinetics of S. aureus in afterglow
argon plasma
Figure 4. Effect of plasma exposure time on germicidal
efficiency (power: 80W; argon flow: 20cm3/min).
The study on germicidal kinetics of plasma is
usually based on the analysis of the specific
characteristics of microorgansm survival curves
resulted from plasma sterilization. Different from the
conventional sterilization, exposure to plasma (be in
direct or in its afterglow zone) provides survival
plots with 3 different linear segments, as shown in
Fig.6. This implies the number of surviving
microorganisms decreases, to a first approximation,
as an exponential function of time also, but with
different time constants, i.e., different kinetics. The
error bars in Fig. 6 indicate the corresponding
standard deviation.
To characterize the slope of each segment, to which
we refer to as an inactivation phase, we use the time
D required to decrease a given population of S.
aureus by a factor of 10 (90 % reduction). Clearly, a
rapid decline in the number of survivors is shown for
initial 30 s (D1) within the distance of 0-30 cm, and
then followed by a gradual drop (D2), finally
followed again by a quick decline (D3). But kill rates
slower with increasing the distance. In contrast,
when the sample was at 60 cm, the results show a
relative tranquilization after 30 s, especially from 30
to 120 s. From these analysis of germicidal kinetics,
and compared with the curves in Fig. 2, we can
obtain the same conclusions as above mentioned,
namely, the charged particles in argon plasma play a
dominant role in the inactivation process but not the
free radicals.
properties of substrate. The intense etching action on
cell materials of S. aureus by charged particles, such
as electrons and ions, plays a dominant role in argon
plasma inactivation process. Exposing S. aureus to
argon plasma (be in direct or in its afterglow zone)
provides survival plots with 3 different linear
segments, i.e., the number of surviving microorganisms decreases as an exponential function of time
but with different kinetics. Within the scope of
effective inactivation (0-30 cm), kill rates slower
with increasing the distance.
Acknowledgment
This work was co-funded (co-supported) by the National
Natural Science Foundation of China (NO. 21077084) and
the Foundational Research Fund of Xi’an Jiaotong
University (NO. 08140009)
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Figure 6. The three-phase survival curves of S. aureus in an
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4. Conclusions
The Decontamination of S. aureus from surfaces by
reduced-pressure afterglow argon plasma was
investigated. Results show that effective inactivation
of S. aureus can be done within the distance of 0-30
cm under the conditions of 100 W power, 240 s
exposure time and 20 cm3/min argon flow. Because
the concentration of electrons and ions is close to
zero and relatively high radicals concentration is
obtained in the afterglow zone ( at about 30 cm from
the active discharge zone), argon plasma sterilization
at distance of 30 cm produce the nicer surface
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