Sterilization Method for Medical Container

Using Microwave-Excited Volume-Wave

Plasma
Masaaki Nagatsu,* Ying Zhao, Iuliana Motrescu, Ryota Mizutani,
Yuya Fujioka, Akihisa Ogino
1. Introduction
Conventionally, steamautoclaves are usedto sterilize heat-
resistant materials andethyleneoxidesterilizers areusedto
sterilize heat-sensitive materials. However, these conven-
tional sterilization and disinfection methods suffer from
various problems. Inparticular, ethylene oxide sterilization
causes environmental problems due to its toxicity. It has
thus been desired to develop a new sterilization technique
that is capable of sterilizingmedical instruments safelyand
rapidly.
Full Paper
M. Nagatsu, Y. Zhao, I. Motrescu, A. Ogino
Graduate School of Science and Technology, Shizuoka University,
3-5-1 Johoku, Naka-ku, Hamamatsu 432-8561, Japan
Fax: 81 53 478 1081; E-mail: tmnagat@ipc.shizuoka.ac.jp
M. Nagatsu, R. Mizutani, Y. Fujioka, A. Ogino
Graduate School of Engineering, Shizuoka University, 3-5-1
Johoku, Naka-ku, Hamamatsu 432-8561, Japan
I. Motrescu
Department of Sciences, The Ion Ionescu De La Brad University
of Agricultural Scienecs and Veterinary Medicine, Aleea M.
Sadoveanu, Iasi 700490, Romania
We demonstrate a novel sterilization technique that sterilizes medical instruments stored in
medical containers by generating a microwave-excited volume-wave plasma inside medical
containers using a planar microwave launcher. We conrmed that a plasma was generated
inside the medical container by the microwaves trans-
mitted through the heat-resistant plastic lid of the
container. A Langmuir probe was used to study the
characteristics of the microwave-excited volume-wave
plasma generated inside the container. The inacti-
vation characteristics of Geobacillus stearothermophi-
lus spores set inside the medical container were also
investigated. 2.3 10
6
spores were inactivated after
irradiation for 40 min or longer by a plasma generated
ina simulated air mixture. This inactivation time could
be reduced to 30 min by adding water vapor to the air-
simulated plasma.
Plasma Process. Polym. 2012, 9, 000000
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com DOI: 10.1002/ppap.201100111 1
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
R
Various plasma sterilization techniques have been devel-
oped that employ low-pressure glow discharge,
[1]
atmo-
spheric-pressure glow discharge,
[24]
downstream plasma
generated by microwave excitation,
[5]
moving atmospheric
microwave plasma
[6]
and surface-wave plasma.
[711]
Plasma sterilization methods have several advantages over
conventional methods. For example, plasmasterilizationcan
be performed at relatively low temperatures and relatively
rapidly. However, plasma sterilization techniques are
generally useful for sterilizing the surfaces of medical
instruments. We recently presented the results of inactiva-
tion measurements of biological indicators (BI) sealed by a
Tyveksheet usingalow-pressuremicrowave-excitedplasma
and showed that 10
6
Geobacillus stearothermophilus spores
were inactivated after irradiation for 60min or longer
without any thermal damage of Tyvek sheet.
[11,12]
In the present study, we describe a novel sterilization
technique in which a plasma is generated inside a medical
container containing medical instruments by microwaves
introduced through a plastic lid using a planar microwave
launcher. Its inactivation properties were investigated
using the spore-forming bacteria, G. stearothermophilus,
which was put inside the medical container together with
the medical instruments.
2. Experimental Section
Todemonstratesterilizationof medical instrumentsinsideamedical
container, we designed and fabricated the prototype microwave
plasma device shown in Figure 1. We used a planar microwave
launcher togenerateamicrowaveplasmainsidethecontainer; it has
been described in detail in previous papers.
[13,14]
In the present
microwave launcher, a quartz disk (diameter: 118mm; thickness:
11mm) was attached to a thin stainless-steel plate by screws (see
Figure 2). We used a metal medical container (length: 27cm; width:
27cm; height: 15cm) that had a plastic lid (Muranaka Medical
Instruments Co.). The lid was made of heat-resistant plastic and
could withstand temperatures up to about 1308C. The body of the
medical container was made of aluminum alloy. The planar
microwavelauncher was attachedtothelidof themedical container
by inserting a silicone rubber sheet as a microwave-transparent
buffer. The discharge gas was introduced into the medical container
through a perforated Tyvek
1
lter tted belowthe plastic lid (black
square in Figure 2b). In the present experiment, we used Ar and a
nitrogen/oxygengas mixture, whichwas used tosimulate air as the
discharge gases. Amicrowave-excited plasma was generated inside
the medical container by introducing microwaves.
To measure the electron density and temperature, a Langmuir
probe witha Cuwire (length: 4mm; diameter: 0.9mm) made witha
semi-rigid cable was inserted in the container through a hermeti-
cally sealed SubMiniature Type A (SMA) connector attached to the
container wall. We used this probe to measure the plasma
parameters inside the container, which was lled with Ar gas.
Sterilization experiments were performed using BIs. The non-
pathogenic spore-forming bacteria, G. stearothermophilus (ATCC#
12980, Raven Biological, US) is commonly used as a sterility
indicator; it contains spore populations in the range of 1.9
2.310
6
. In the present study, its spores were pasted on a small
rectangular stainless-steel (SUS) plate, which was placed in a
Tyvek
1
/polypouch. For colony forming unit (CFU) count, BIs that
had not been exposed to plasma were used as a control. Spore
survivors fromboththeplasma-exposedandthecontrol BI samples
were recovered by plunging the carriers into 1.5ml of brainheart
infusionsolutioninatest tube. Test tubes containingtheBI carriers
were vortexed for 1 min at room temperature. 0.1ml of the spore
suspension from the test tube was inoculated onto nutrient agar
media with triple replication. The survivors were counted as the
number of CFU per BI carrier after incubation at 55 8C for 24 h.
Survival curves were obtained by plotting the CFU counts results.
Another simple way to evaluate the inactivationof spores is to use
tryptic soybroth(TSB) as a culture solutionandbromocresol purple
as a pHindicator. If spores survive, the color of the culture solution
changes from purple to yellow after incubation for 24h.
3. Results and Discussion
3.1. Discharge Characteristics of Microwave Plasma
Generated in the Container
To conrm the plasma generation below the plastic lid
attached by a planar microwave launcher, we carried out
Figure 1. Photograph and schematic diagram of prototype micro-
wave plasma device.
2
Plasma Process. Polym. 2012, 9, 000000
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ppap.201100111
R
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
M. Nagatsu, Y. Zhao, I. Motrescu, R. Mizutani, Y. Fujioka, A. Ogino
the experiments shown in Figure 3. We compared the
plasma discharges with and without the plastic lid of the
container belowthe planar microwave launcher, as shown
inFigure 3a. Photographs of plasma discharges with Ar and
air as working at gas pressure of 6.7 Pa and gas owrate of
70 sccmgas are shown in Figure 3b and c, respectively. The
incident microwave power was about 300W and reected
one was roughly 2030 W. The electron density in the case
of Ar plasma was measured by the Langmuir probe located
at 5.5cmbelowthe bottomsurface of microwave launcher.
In the case without the lid, the electron density was about
2.5 10
11
cm
3
, which was higher than the cutoff density,
7.4 10
10
cm
3
. It is expectedthat the electrondensitynear
the microwave launcher will be higher than the critical
density of surface-wave, 3.6 10
11
cm
3
. This condition is
generally satised in a typical surface-wave plasma
generation. On the other hand, in the case with the lid,
the density was 5610
10
cm
3
by a factor of 45 lower
than the case without the lid, that is, lower than the cutoff
density. Such plasma is characteristic of the volume-wave
plasma.
[15]
When we installed the medical container below the
microwave launcher witharubber buffer sheet inbetween,
microwaves propagated through the plastic lid into the
container where they generated a plasma. The plasma
discharge inside the medical container
could be easily conrmed by using a
medical container with a metal mesh
sidewall. Figure 4 shows photographs
taken before and after turning on the Ar
plasma discharge in the medical con-
tainer. It also contains a schematic
drawing of the container with the mesh
sidewall. The plasma was generated
using a microwave power of 400W at
an Ar gas pressure of 27 Pa and a gas ow
rate of 200 sccm.
We performed Langmuir probe mea-
surements to determine whether a sur-
face-wave or volume-wave plasma was
generated inside the container. The posi-
tion of the probe tip was varied from
horizontal to vertical by bending the
cable. Figure 5 depicts the probe mea-
surement geometry. We dened the
center of the container as r 0 and the
axial (vertical) position z was measured
relative to the bottom of the container,
which was dened as z 0. The diagonal
direction is dened as the projection of
the r axis onto the horizontal plane, as
illustrated in Figure 5.
As mentioned above, surface-wave
excitation by 2.45 GHz microwaves
Figure 3. (a) Schematics of experimental geometries and photographs of plasma
discharges generated by the planar microwave launcher attached with (right) and
without (left) a plastic lid of container in the cases of (b) Ar and (c) air, respectively.
Figure 2. Photographs of (a) planar microwave launcher and
(b) medical container.
Plasma Process. Polym. 2012, 9, 000000
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plasma-polymers.org 3
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
R
Sterilization Method for Medical Container
requires a plasma density higher than the critical density
(3.6 10
11
cm
3
), whereas volume-wave plasmas are
generated at plasma densities below the cutoff density
(7.4 10
10
cm
3
).
[15]
Ar plasmas were generated at
various microwave powers in the range 150400 W at a
pressure of 40 Pa and a gas ow rate of 100 sccm. Figure 6
shows the dependence of the electron density and electron
temperature on the total incident microwave power when
the probe was located at r 0 and z 6 cm. The electron
temperature is almost constant at about 1.5eV at different
microwave powers, whereas the electron density increases
approximatelylinearlywiththeincident microwavepower
and it appears to be less than the cutoff density. These
results suggest that the plasma generated inside the
container is probably a volume-wave plasma. Figure 7
shows the electron density distributions along the r-axis at
z 6and8cm, whichweremeasuredbyscanningtheprobe
in the radial direction. The total incident microwave power
was 300W, the Ar gas pressure was 40 Pa, and the gas ow
rate was 100sccm. At z 8cm, where the probe is near the
lid, theelectrondensitypeakedat thecenter sinceavolume-
wave plasmawas generatedimmediatelybelowthe plastic
lid, which was beneath the microwave launcher. In
contrast, the plasma extends to the container wall at
z 6 cm and thus the electron density prole is broader
thanthat at z 8cm. Thenext sectionconsiders theeffect of
a plasma generated in simulated air mixture on the
inactivation of BIs.
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12 14
E
l
e
c
t
r
o
n

D
e
n
s
i
t
y

(
x
1
0
9

c
m
-
3
)
Distance from the Center r (cm)
z=8 cm
z=6 cm
Figure 7. Electron density distributions along the r axis at z 6cm
and z 8cm (total incident power: 300W; Ar gas pressure: 40Pa;
gas ow rate: 100sccm).
Figure 4. Schematic diagram of medical container with an open-
structured, mesh sidewall, and photographs before and after Ar
plasma discharge inside the medical container.
Figure 5. Geometry of Langmuir probe measurements.
0
0.5
1
1.5
2
2.5
0
2
4
6
8
10
150 200 250 300 350 400
E
l
e
c
t
r
o
n

T
e
m
p
e
r
a
t
u
r
e

(
e
V
)
E
l
e
c
t
r
o
n

D
e
n
s
i
t
y

(

x

1
0
9

c
m
-
3
)
Total Incident Power (W)
Figure 6. Dependence of electron density and electron tempera-
ture at a probe position of r 0 and z 6cm on the total incident
microwave power.
4
Plasma Process. Polym. 2012, 9, 000000
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ppap.201100111
R
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
M. Nagatsu, Y. Zhao, I. Motrescu, R. Mizutani, Y. Fujioka, A. Ogino
3.2. Inactivation of BIs Inside the Container by a
Volume-Wave Plasma
In an experiment to investigate the inactivation of BIs set
inside the container, we employed a simulated air mixture
of nitrogen(160 sccm) andoxygen(40 sccm) at apressure of
about 90 Pa instead of Ar gas. Amicrowave-excited plasma
was generated using time-modulated microwaves with an
on-time of 30 s and an off-time of 60 s to prevent thermal
damage to the plastic lid of the container; the total
microwave power was about 300W. We rst investigated
the lethal properties of the microwave-excited volume-
wave plasma on BIs by mounting a SUS mesh basket inside
the container (see Figure 8). Total plasma irradiation times
of 10, 20, 30 and 40 min were used. To investigate the
inactivation properties at different positions, we set the BIs
at three different positions: in the left corner (BI-1), in the
center (BI-2), and in the right corner (BI-3) (see Figure 8).
These BIs are located at about z 6 cm. We used heat-
sensitive labels (Thermo Label 5E-75, 5E-100 and 5E-125,
Nichiyu) to monitor the temperature near the sample
position and the back of the plastic lid. Table 1 shows
the results obtained for plasma inactivation of
G. stearothermophilus using a TSB culture solution with
bromocresol purple. After plasma treatment, the samples
were incubated at 5560 8C for more than 1 d, which is
standard for G. stearothermophilus. The spore mortality
was monitored by daily checking the color of the TSB
solution during incubation. Figure 9 shows the survival
curves obtained for G. stearothermophilus at the center and
near the edge of the container. They show that BIs were
inactivated after 40 min of plasma treatment. The heat-
sensitive labels show that the temperature at the back of
the lid in the center of the container was 75 8C<T <80 8C
and that near the BI-2 samples was 95 8C<T <1008C after
irradiation for 40 min.
According to the previous results,
[11,12,16]
we consider
that the main mechanism of bacterial inactivation is the
synergetic effect of the etching of bacteria due to oxygen
radicals and VUV/UV emission by O atoms, N atoms, NO
molecules, andN
2
molecules excitedinthe air plasma. From
the morphology analysis using the scanning electron
microscopy, it was found that the spores were signicantly
eroded by the excited O atoms, which leads to the fatal
inactivation of spores.
In a previous paper,
[12]
we reported the effect of addition
of water vapor to the simulated air mixture of nitrogenand
oxygengases onthesterilitycharacteristics of BIs. Wefound
that the addition of a small amount of water vapor caused
G. stearothermophilus spores to become inactivated faster
than when a dry air gas plasma was used. To demonstrate
the effect of the addition of water vapor on the sterilization
of the interior of the medical container, we investigatedthe
effect of adding water vapor to the simulated air gas
mixture. Table 2 and Figure 10, respectively, show the
results for TSB culture solution tests and colony counting
methods. Both results indicate that 10
6
BI spores were
inactivatedapproximately10 minfaster whenwater vapor
Table 1. Results for inactivation of G. stearothermophilus in a TSB
culture solution by a volume-wave plasma generated in a simu-
lated-air mixture.
Treatment
time
[min]
BI-1
(left corner)
BI-2
(center)
BI-3
(right corner)
10
20
30
40
10
-1
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
0 10 20 30 40 50
G.stearothermophilus
N
2
/O
2
160/40sccm
%,
%,
%,
C
o
l
o
n
y

F
o
r
m
i
n
g

U
n
i
t
s

(
C
F
U
)
Plasma Treatment Time [min]
Bl-2
Bl-1
Bl-3
G. stearothermophilus
N
2
/O
2
160/40 sccm
Figure 9. Survival curves for G. stearothermophilus at center and
near the edge after irradiation by a plasma in a simulated air
mixture.
Figure 8. Photograph of inside of container containing SUS mesh
basket indicating the three locations of BIs.
Plasma Process. Polym. 2012, 9, 000000
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plasma-polymers.org 5
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
R
Sterilization Method for Medical Container
was added at a partial pressure of 6.8 Pa relative to whenno
vapor was added(seeTable1andFigure9). This reductionin
the inactivation time may be due to OH radicals produced
from the added water vapor acting as strong oxidizing
radicals in the inactivation process.
Finally, tosimulate arealistic situation, we measuredthe
inactivation properties of BIs by placing medical instru-
ments, such as forceps, surgical knifes, and tweezers, in the
stainless-steel mesh basket (see Figure 11). Although these
metallic instruments were inside the container, a plasma
could still be easily generated using the same discharge
conditions as previously. Figure 12 shows survival curves
for BIs at three different positions and roughly z 8 cm.
They are similar to those obtained when an empty metal
mesh basket was set inside the container. The temperature
after treatment for 40 min was 80 8C<T <85 8C at the back
of the lid and 75 8C<T <80 8C near the BIs located in the
center. These temperatures are slightly lower than those
obtained when no medical instruments were present. This
may be because the high thermal conductivities of the
metallic medical instruments cause them to act heat sinks
during plasma treatment.
Here, to discuss the heat effect on the inactivation of BIs,
we carried out a simple experiment in the atmosphere,
where the BIs were put on the temperature-controlled hot
plate, where the temperature was kept at 70 and 100 8C
withinaripple of 1 8C. Figure 13shows the survival curves
of G. stearothermophilus treatedat 70and100 8C. Evenafter
40 min at 1008C, roughly 10
6
spores were still surviving.
Table 2. Results for inactivation of G. stearothermophilus in a TSB
culture solution by a volume-wave plasma generated in a simu-
lated-air mixture to which water vapor had been added.
Treatment
time
[min]
BI-1
(left corner)
BI-2
(center)
BI-3
(right corner)
10
20
30
40
10
-1
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
0 10 20 30 40 50
G.stearothermophilus
N
2
/O
2
160/40sccm
water 75000ppm
%,
%,
%,
C
o
l
o
n
y

F
o
r
m
i
n
g

U
n
i
t
s
(
C
F
U
)
Bl-1
Bl-3
Bl-2
wi th water vapor
Plasma Treatment Time (min)
G. stearothermophilus
N
2
/O
2
160/40 sccm
with water vapor
Figure 10. Survival curves for G. stearothermophilus at center and
near the edge after irradiation by a plasma in a simulated air
mixture containing water vapor.
Figure 11. Photograph of inside of container containing SUS mesh
basket lled with medical instruments indicating the three
locations of BIs.
Figure 12. Survival curves for G. stearothermophilus at center and
near the edge after irradiation by a plasma in a simulated air
mixture containing water vapor when the basket contains
medical instruments.
6
Plasma Process. Polym. 2012, 9, 000000
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ppap.201100111
R
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
M. Nagatsu, Y. Zhao, I. Motrescu, R. Mizutani, Y. Fujioka, A. Ogino
This indicates that the heat effect on spore inactivation is
negligibly small in the proposed sterilization method.
4. Conclusion
A novel technique for generating a stable microwave-
excited plasma inside a medical container is proposed.
Using this technique, medical instruments in a medical
container can be readily sterilized without opening the
container, just like sterilization using an autoclave.
The present experiments conrmed that a plasma could
be generated inside a medical container by transmitting
microwaves through the heat-resistant plastic lid of the
container. The characteristics of microwave-excited
volume-wave plasma generated in the container were
studied using a Langmuir probe. G. stearothermophilus
witha populationof 2.310
6
ina metal meshbasket inthe
medical container became inactivatedafter plasma irradia-
tion for 40 min or longer by using a plasma in an air-
simulated mixture with a gas ow of 200sccm when the
total microwave power was 300 W and the pressure was
90 Pa. This was true even when medical instruments were
placedinthebasket insidethecontainer. Whenwater vapor
was added, the BIs were inactivated about 10 min quicker
than when dry simulated air was used.
The present experimental results demonstrate rapid
(<30 min) sterilizationof the interior of amedical container
by a microwave-excited volume-wave plasma at a rela-
tively low temperature (<1008C).
Acknowledgements: This work was partly supported by a grant-
in-aid for Scientic Research from the Japan Society for the
Promotion of Science (JSPS).
Received: June 5, 2011; Revised: October 15, 2011; Accepted:
October 24, 2011; DOI: 10.1002/ppap.201100111
Keywords: low-pressure discharges; microwave discharges;
spores; sterilization
[1] I. A. Soloshenko, V. V. Tsiolko, V. A. Khomich, A. I. Shchedrin,
A. V. Ryabtsev, V. Yu. Bazhenov, I. L. Mikhno, Plasma Phys. Rep.
2000, 26, 792.
[2] T. C. Montie, K. Kelly-Wintenberg, J. R. Roth, IEEE Trans.
Plasma Sci. 2000, 28, 41.
[3] M. Laroussi, I. Alexeff, W. L. Kang, IEEE Trans. Plasma Sci. 2000,
28, 184.
[4] V. Y. Bazhenov, A. I. Kuzmichev, V. I. Kryzhanovsky, I. L.
Mikhno, A. V. Ryabtsev, I. A. Soloshenko, V. A. Khomich,
V. V. Tsiolko, A. I. Shchedrin, Proc. 15th Int. Symp. Plasma
Chem. Orleans, France, Vol. II, 2001, 3005.
[5] M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, M. Tabriziani, L.
H. Yahia, Int. J. Pharm. 2001, 226, 1.
[6] J. Ehlbeck, A. Ohl, M. Maas, U. Krohmann, T. Neumann, Surf.
Coat. Technol. 2003, 174175, 493.
[7] S. Lerouge, M. R. Wertheimer, R. Marchand, M. Tabriziani, L. H.
Yahia, J. Biomed. Mater. Res. 2000, 51, 128.
[8] M. Nagatsu, F. Terashita, Y. Koide, Jpn. J. Appl. Phys. 2003, 42,
L856.
[9] M. Nagatsu, F. Terashita, H. Nonaka, L. Xu, T. Nagata, Y. Koide,
Appl. Phys. Lett. 2005, 86, 211502.
[10] J. Pollak, M. Moisan, D. Keroack, M. K. Boudam, J. Phys. D:
Appl. Phys. 2008, 41, 135212.
[11] M. K. Singh, A. Ogino, M. Nagatsu, New J. Phys. 2009, 11,
115027.
[12] M. K. Singh, A. Ogino, M. Nagatsu, L. Xu, J. Plasma Fus. Res. Ser.
2009, 8, 560.
[13] M. Nagatsu, K. Naito, A. Ogino, K. Ninomiya, S. Nanko, Appl.
Phys. Lett. 2005, 87, 161501.
[14] M. Nagatsu, K. Naito, A. Ogino, S. Nanko, Plasma Sources Sci.
Technol. 2006, 15, 37.
[15] A. Ogino, K. Naito, F. Terashita, S. Nanko, M. Nagatsu, Jpn. J.
Appl. Phys. 2005, 44, L352.
[16] Y. Zhao, A. Ogino, M. Nagatsu, Appl. Phys. Lett. 2011, 98,
191501.
10
-1
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
0 10 20 30 40 50
C
o
l
o
n
y

F
o
r
m
i
n
g

U
n
i
t
s
Treatment time (min)
70 degC
100 degC
Figure 13. Survival curves for G. stearothermophilus heated at 70
and 1008C.
Plasma Process. Polym. 2012, 9, 000000
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plasma-polymers.org 7
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
R
Sterilization Method for Medical Container