Plasma Process Polym Art

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Sterilization Method for Medical ContainerUsing Microwave-Excited Volume-Wave

Plasma

Masaaki Nagatsu,* Ying Zhao, Iuliana Motrescu, Ryota Mizutani,Yuya Fujioka, Akihisa Ogino

1. Introduction

Conventionally, steamautoclaves are used to sterilizeheat-

resistantmaterialsandethyleneoxidesterilizersareusedto

sterilize heat-sensitive materials. However, these conven-

tional sterilization and disinfection methods suffer from

various problems. In particular,ethylene oxidesterilization

causes environmental problems due to its toxicity. It has

thus been desired to develop a new sterilization technique

thatis capable of sterilizing medical 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: þ81 53 478 1081; E-mail: [email protected]

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 confirmed 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 Â 106 spores were inactivated after

irradiation for 40 min or longer by a plasma generated

in a 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 , 000–000ß 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,[2–4] downstream plasma

generated by microwave excitation,[5] moving atmospheric

microwave plasma[6] and surface-wave plasma.[7–11]

Plasma sterilization methods have several advantages overconventionalmethods.Forexample,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

Tyveksheetusingalow-pressuremicrowave-excitedplasma

and showed that 106 Geobacillus stearothermophilus spores

were inactivated after irradiation for 60 min 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 microwavesintroduced 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

Todemonstratesterilizationofmedicalinstrumentsinsideamedical

container, we designed and fabricated the prototype microwave

plasma device shown in Figure 1. We used a planar microwave

launcher togenerate a microwave plasmainsidethecontainer;it hasbeen described in detail in previous papers.[13,14] In the present

microwave launcher, a quartz disk (diameter: 118 mm; thickness:

11 mm) 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

microwavelauncherwasattachedtothelidofthemedicalcontainer

by inserting a silicone rubber sheet as a microwave-transparent

buffer.The discharge gaswas introduced intothe medical container

througha perforated Tyvek1filterfitted below the plastic lid(black

square in Figure 2b). In the present experiment, we used Ar and a

nitrogen/oxygengas mixture, which wasused to simulateair as the

discharge gases. A microwave-excited plasma was generated inside

the medical container by introducing microwaves.

To measure the electron density and temperature, a Langmuir

probe witha Cuwire(length: 4 mm;diameter: 0.9 mm) 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 filled 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 1.9–2.3 Â 106. In the present study, its spores were pasted on a small

rectangular stainless-steel (SUS) plate, which was placed in a

Tyvek1/polypouch. For colony forming unit (CFU) count, BIs that

had not been exposed to plasma were used as a control. Spore

survivorsfromboththeplasma-exposedandthecontrolBIsamples

were recovered by plunging the carriers into 1.5ml of brain–heart

infusionsolutionin a test tube. Test tubescontaining theBI carriers

were vortexed for 1 min at room temperature. 0.1 ml 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 24h.

Survival curves were obtained by plotting the CFU counts results.

Another simpleway to evaluate theinactivation of spores is to use

tryptic soybroth (TSB)as a culture solutionand bromocresolpurpleas a pH indicator. If sporessurvive,the color of theculture solution

changes from purple to yellow after incubation for 24 h.

3. Results and Discussion

3.1. Discharge Characteristics of Microwave Plasma

Generated in the Container

To confirm 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.

2Plasma Process. Polym. 2012 , 9, 000–000ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ppap.201100111

REarly 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 below the planar microwave launcher, as shown

in Figure 3a.Photographs of plasma discharges with Ar and

air as working at gas pressure of 6.7 Pa and gas flow rate of

70sccm gas are shown in Figure 3b and c, respectively. Theincident microwave power was about 300W and reflected

one was roughly 20–30 W. The electron density in the case

of Ar plasma was measured by the Langmuir probe located

at 5.5cm below the bottomsurface of microwave launcher.

In the case without the lid, the electron density was about

2.5 Â 1011 cmÀ3, which was higher than the cutoff density,

7.4 Â 1010 cmÀ3. It is expected that theelectrondensitynear

the microwave launcher will be higher than the critical

density of surface-wave, 3.6 Â 1011 cmÀ3. This condition is

generally satisfied in a typical surface-wave plasma

generation. On the other hand, in the case with the lid,

the density was 5–6 Â 1010 cmÀ3 by a factor of 4–5 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

microwavelauncherwith a rubber buffersheetin between,

microwaves propagated through the plastic lid into the

container where they generated a plasma. The plasma

discharge inside the medical container

could be easily confirmed 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 400 W at

anAr gas pressureof 27Pa and a gas flow

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 defined the

center of the container as r ¼ 0 and the

axial (vertical) position z was measured

relative to the bottom of the container,

which was defined as z ¼ 0. The diagonal

direction is defined as the projection of

the r axis onto the horizontal plane, as

illustrated in Figure 5.

As mentioned above, surface-wave

excitation by 2.45GHz microwaves

Figure 3. (a) Schematics of experimental geometries and photographs of plasmadischarges generated by the planar microwave launcher attached with (right) andwithout (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 , 000–000ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plasma-polymers.org 3


Sterilization Method for Medical Container



requires a plasma density higher than the critical density

(¼ 3.6 Â 1011 cmÀ3), whereas volume-wave plasmas are

generated at plasma densities below the cutoff density

(¼ 7.4 Â 1010 cmÀ3).[15] Ar plasmas were generated at

various microwave powers in the range 150–400 W at a

pressure of 40 Pa and a gas flow 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 ¼ 0 and z ¼ 6cm. The electron

temperature is almost constant at about 1.5eV at different

microwave powers, whereas the electron density increases

approximatelylinearlywiththeincidentmicrowavepower

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 ¼ 6 and8 cm, whichwere measured byscanningthe probe

in the radial direction. The total incident microwave power

was 300 W, the Ar gas pressure was 40Pa, and the gas flow

rate was 100 sccm. At z ¼ 8 cm, where the probe is near the

lid, theelectron density peakedat thecentersincea volume-

wave plasma was generated immediately belowthe plastic

lid, which was beneath the microwave launcher. In

contrast, the plasma extends to the container wall at

z ¼ 6 cm and thus the electron density profile is broader

thanthatat z ¼ 8cm.Thenextsectionconsiderstheeffectof

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 ¼ 6 cmand z ¼ 8 cm (total incident power: 300 W; Ar gas pressure: 40 Pa;gas flow rate: 100sccm).

Figure 4. Schematic diagram of medical container with an open-structured, mesh sidewall, and photographs before and after Arplasma 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 ¼ 0 and z ¼ 6 cm on the total incidentmicrowave power.






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 (40sccm) at a pressure of

about 90 Pa instead of Ar gas. A microwave-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 300 W. We first 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

inactivationproperties at differentpositions, 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 ¼ 6cm. 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 55–60 8C for more than 1d, 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 958C<T <100

8C 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,andN2moleculesexcitedin theair plasma. From

the morphology analysis using the scanning electron

microscopy, it was found that the spores were significantly

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 thesimulated air mixture of nitrogen and

oxygengasesonthesterilitycharacteristicsofBIs.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 theinteriorof 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 106 BI spores were

inactivated approximately 10 minfaster when watervapor

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 þ þ þ

20 þ þ þ

30 þ – þ

40 – – –

10-1

100

101

102

103

10

4

105

106

107

0 10 20 30 40 50

G.stearothermophilus

N2 /O

2160/40sccm

%,%,%,

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]

%,

%,

%,

G. stearothermophilus

N2/O2 160/40 sccm

Figure 9. Survival curves for G. stearothermophilus at center and

near the edge after irradiation by a plasma in a simulated airmixture.

Figure 8. Photograph of inside of container containing SUS meshbasket indicating the three locations of BIs.






was added ata partial pressureof 6.8 Parelative towhenno

vaporwasadded(seeTable1andFigure9).Thisreductionin

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, to simulatea realisticsituation, we measured the

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 ¼ 8cm.

They are similar to those obtained when an empty metal

mesh basket was set inside the container. The temperature

after treatment for 40min was 80 8C<T <85 8C attheback

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

withinarippleof Æ1 8C. Figure13 shows thesurvivalcurves

of G. stearothermophilus treatedat70and100 8C.Evenafter

40 min at 100 8C, roughly 106 spores were still surviving.

Table 2. Results for inactivation of G. stearothermophilus in a TSBculture 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 þ þ þ

20 þ – þ

30 – – –

40 – – –

10-1

100

101

102

103

104

10

5

106

107

0 10 20 30 40 50

G.stearothermophilus

N2 /O

2160/40sccm

water 75000ppm

%,%,%,

C o l o n y F o r m i n g U n i t s ( C

F U )

%,

%,

%,

ZLWKZDWHUYDSRU

Plasma Treatment Time (min )

G. stearothermophilus

N2/O2 160/40 sccm

with water vapor

Figure 10. Survival curves for G. stearothermophilus at center andnear the edge after irradiation by a plasma in a simulated airmixture containing water vapor.

Figure 11. Photograph of inside of container containing SUS meshbasket filled with medical instruments indicating the threelocations of BIs.

Figure 12. Survival curves for G. stearothermophilus at center andnear the edge after irradiation by a plasma in a simulated airmixture containing water vapor when the basket containsmedical instruments.






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 confirmed 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

with a population of 2.3 Â 106 inametalmeshbasketinthe

medical containerbecame inactivated afterplasma irradia-

tion for 40min or longer by using a plasma in an air-

simulated mixture with a gas flow of 200 sccm when the

total microwave power was 300 W and the pressure was

90 Pa. This was true even when medical instruments were

placedin thebasketinside thecontainer.Whenwatervaporwas added, the BIs were inactivated about 10 min quicker

than when dry simulated air was used.

The present experimental results demonstrate rapid

(<30 min) sterilization of theinteriorof a medical container

by a microwave-excited volume-wave plasma at a rela-

tively low temperature (<100 8C).

Acknowledgements: This work was partly supported by a grant-in-aid for Scientific Research from the Japan Society for thePromotion 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

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10-1

100

101

102

103

10

4

105

106

107

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 70and 100 8C.




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