Bacterial Inactivation Using a Low-Temperature Atmospheric

Plasma Brush Sustained With Argon Gas

Q. S. Yu,1 C. Huang,1 F.-H. Hsieh,2 H. Huff,2 Yixiang Duan3
1

Center for Surface Science and Plasma Technology and Department of Chemical Engineering, University of
Missouri-Columbia, Columbia, Missouri 65211
2

Department of Biological Engineering, University of Missouri-Columbia, Columbia, Missouri 65211

Los Alamos National Laboratory, Los Alamos, New Mexico 87545

Received 18 August 2005; revised 27 January 2006; accepted 22 February 2006

Published online 18 July 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30586

Abstract: This study investigated the bacterial inactivation/sterilization effects of a new

atmospheric plasma source, which is a brush-shaped argon glow discharge created under 1
atm pressure. Such an atmospheric plasma brush requires extremely low power of less than
20 W to operate; and therefore is essentially a low-temperature discharge as conrmed by
gas-phase temperature measurements. Two bacteria, Escherichia coli (E. coli) and Micrococcus luteus (M. luteus), seeded in various media were subjected to plasma treatment and their
survivability was examined. It was found that such argon atmospheric plasma brush is very
effective in destruction of the bacteria cells. With nutrient broth and standard methods agar
as supporting media, a cell reduction in a level of 6 orders of magnitude was observed for E.
coli within 3 4 min plasma treatment. A similar level of cell reduction was also observed for
M. luteus in the two media with 2 or 3 min plasma treatment. The plasma treatment effects on
the bacteria cell structures were also examined using scanning electron microscopy and the
cell structure damages due to the plasma exposure were observed on both bacteria. The
possible sterilization mechanism of the argon plasmas is also discussed in this article. 2006
Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 80B: 211219, 2007

Keywords:
luteus

atmospheric plasma; glow discharge; sterilization; Escherichia coli; Micrococcus

INTRODUCTION
Sterilization is a process that is used to destroy unwanted
microorganisms on the surface of a member. Conventional
sterilization methods may be categorized into heat sterilization (use of saturated pressurized steam, autoclaving or dry
heat), gaseous chemical sterilization (using ethylene oxide,
EtO, ozone, orformaldehyde), irradiation sterilization (using
electromagnetic, particle, ultraviolet irradiation source), and
ltration sterilization methods.1,2 Each of these conventional
sterilization methods has its own set of advantages and disadvantages. Heat sterilization is usually employed to kill
bacteria, viable spores, and viruses in heat resistant materials.
But heat sterilization is an extremely time-consuming process
and is not suitable for articles made of heat sensitive materials
such as plastics and fabrics. Gaseous chemical sterilization is

Correspondence to: Q. S. Yu (e-mail: yuq@missouri.edu)

Contact grant sponsor: College of Engineering, University of Missouri-Columbia
Contact grant sponsor: Bioprocessing and Biosensing Center, University of
Missouri-Columbia
2006 Wiley Periodicals, Inc.

capable of sterilizing at low temperature but is limited by its

use of highly toxic gases. Irradiation sterilization may cause
undesirable changes in materials and must be used with
caution to avoid dispersing problem. Both chemical sterilization and irradiation sterilization require professionally welltrained personnel to operate, and the cost of operation is high.
Strictly speaking, ltration is not a sterilization process because it cannot destroy or remove all microorganisms. For
these reasons, there is a need to develop a safe, rapid, less
expensive, environmental friendly sterilization method to
eliminate the drawbacks of the conventional sterilization processes.3
For sterilization of biomedical devices, low-temperature/
low-pressure plasma technique, which utilizes reactive species (such as free atoms and energetic electrons) contained in
the plasma, has been used as an alternative to the conventional sterilization methods discussed previously.4 Although
this plasma technique has a high efciency, it suffers from a
number of drawbacks, such as being limited to low-pressure
plasma, having a restricted plasma reactor volume, and the
need for several vacuum cycles.
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YU ET AL.

Since the late 1980s, various nonthermal atmospheric

pressure plasma sources have been developed. These plasmas
can be created in different forms, including dielectric barrier
discharge,57 corona discharge,8 resistive discharge,9 11 and
atmospheric pressure plasma jets sustained by radio frequency or microwave.12,13 Since atmospheric plasma is operated at 1 atm pressure, it eliminates the need for vacuum
equipment and thus signicantly reduces the complexity and
the operational cost of the system. These nonthermal atmospheric plasmas do possess some of the reactive characteristics of low-pressure plasmas. It has been shown that when
these plasmas are used for sterilization, they do not cause
pain or bulk destruction of living tissue,14,15 do not damage
heat-sensitive materials, and are as safe as EtO.16 Therefore,
low-temperature plasma sterilization is a viable method for
use in biomedical applications. However, the atmospheric
plasma sources in current use suffer from the plasma volume
or size being limited by the electrode spacing of the discharge. Plasma jets have the potential for increasing the
volume of material sterilized but the high power consumption
increases the process cost substantially and may damage the
surface of many materials.
In the present study, we present a new plasma source, i.e.,
a low-temperature atmospheric plasma brush ignited and
sustained at very low power consumption. We investigated
the efciency of a method, in which this plasma brush was
operated with argon gas, for destroying Escherichia coli (E.
coli) and Micrococcus luteus (M. luteus) on various supporting media.

Figure 2. Temperature change of argon atmospheric plasmas with

argon ow rate at different dc power input.

General Store of University of Missouri-Columbia. The bacteria, E. coli and M. luteus, were obtained from the Department of Food Science, University of Missouri-Columbia. The
qualitative P5 lter paper (11.0 cm in diameter) used as the
porous solid medium for the bacteria was obtained from
Fisher Scientic (Houston, TX). The nutrient broth (pH 6.8 at
25C) was purchased from Difco Bacto (Detroit, MI), and the
standard methods agar was purchased from Becton Dickson
(Cockeysville, MD).
Low-Temperature Atmospheric Plasma Brush

MATERIALS AND METHODS

Materials and Organisms

The gas used to create the atmospheric plasma was an industrial grade argon with 99.997% purity and purchased from

The design details of the discharge chamber for the plasma

source are proprietary. The argon gas passes through the
discharge chamber at a ow rate controlled by a MKS mass
ow controller (MKS Instruments Andover, MA). An electrical eld was applied to the two electrodes located inside the
chamber to ignite a dc glow discharge by a dc power supply
(Pd 1556C, Power Design New York, NY). Figure 1 shows
the pictorial view of the low-temperature atmospheric plasma
brush that touches a paper surface.
This plasma source can be operated under very low electrical power (as low as a few watts), and as a result very low
plasma temperature can be achieved. The gas phase temperatures of such an argon atmospheric plasma, measured using
a thermocouple thermometer (Fisherbrand Traceable Dual
Channel Thermometer, Fisher Scientic, Houston, TX) range
from 40 to 160C, with the corresponding argon gas ow rate
being from 3500 sccm (standard cubic centimeter per minute,
a volumetric ow rate at 273 K under 1 atm) to 1000 sccm
(Figure 2).
Experimental Procedures

Figure 1. Photographs of argon atmospheric plasmas touching a

paper surface. Plasma conditions were 1500 sccm argon, 15 W dc
power input. [Color gure can be viewed in the online issue, which is
available at www.interscience.wiley.com.]

The E. coli or M. luteus organisms in the stock nutrient broth

solution were kept at 35C in an incubator. Prior to the
plasma treatment, the bacteria were transferred into 10 mL
nutrient broth and allowed to grow for 24 h. All experiments
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213

Figure 3. Survival curves of (A) Escherichia coli and (B) Micrococcus luteus in various supporting
media after argon atmospheric plasma treatment with argon ow rate of 1500 sccm and dc power
input of 15 W. [Color gure can be viewed in the online issue, which is available at www.
interscience.wiley.com.]

were done in parallel with two specimens and the plating was
done in duplicate for each of the two specimens unless
otherwise noted. The values reported for cell reduction are
the average of the results obtained from two specimens.

tone water and mixed using a vortex mixer for 2 min. The
bacterial strains were serially diluted and dispersed into the
agar in petri dishes. The number of the living bacteria cells
was counted after incubation at 35C for 48 h.

Bacteria on Filter Papers (Porous Solid Medium)

Bacteria in Nutrient Broth (Liquid Medium)

With lter paper as the media, the 5 L nutrient broth

containing the bacteria were dispensed onto sterilized lter
paper discs (4 mm diameter), and the discs were then allowed
to dry in a desiccator for 1 h. The lter paper discs containing
bacteria were treated with the low-temperature atmospheric
plasma brush with a preset exposure time from 1.0 to 5.0 min.
After the plasma treatment, the plasma treated lter paper
discs were transferred into test tubes containing 10 mL pep-

Nutrient broth (5 L) containing the bacteria was dispensed

on a sterilized Al dish (15 mm in diameter) to form a liquid
droplet of 1.0 mm in diameter. The droplet was then placed
under the atmospheric plasma brush for the plasma treatment.
At the end of the treatment, the Al dish was washed two times
with peptone water, and the remaining bacteria strains were
transferred into test tubes that contained 10 mL peptone
water. The solution was then mixed for 2 min using a vortex

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YU ET AL.

Figure 4. The surviving cell numbers of Escherichia coli and Micrococcus luteus on lter papers as a
function of plasma parameters: (A) plasma power and (B) argon ow rate after 1 min plasma treatment
under conditions, if not specied in the plots, of 1500 sccm argon and 15 W dc power input.

mixer and then serially diluted and dispersed into the agar in
petri dishes. The number of the living bacteria cells was
counted after incubation at 35C for 48 h.
Bacteria in Standard Methods Agar (Colloid Medium)

Nutrient broth (5 L) containing the bacteria was mixed with

30 L standard methods agar in a sterilized Al dish (15 mm
in diameter) and allowed to harden to form agar plug (3 mm
in thickness and 5 mm in diameter). The agar plug on the
sterilized Al dishes was then placed under the atmospheric
plasma brush for the plasma treatment. At the end of the
treatment, the dishes were macerated two times and then
washed with peptone water. The solution was then mixed for
2 min using a vortex mixer and serially diluted and dispersed
into the agar in petri dishes. The number of the living bacteria
cells was counted after incubation at 35C for 48 h.

Effect of Position of the Plasma

To examine the sterilization effects of position of the plasma,

the bacteria seeded in lter papers were subjected to plasma
exposure both inside the plasma glow and in the down stream
at a position 2 mm away from the visible glow. In such a
remote plasma position, only the long-life plasma species
from the plasmas were allowed to diffuse and got in contact
with the bacteria cells that resided on the supporting media.
Effects of Dry Heat and Gas Blowing on Cell Reduction

In order to distinguish the plasma sterilization effects from

the possible heat effect of the plasmas, a sterilization test to
elucidate the dry heat effect was performed using a temperature controllable oven (Fisher Scientic Isotemp Vacuum
Oven Model 282 A). Under the operating condition studied,
the gas phase temperature of the argon plasmas was 125C.
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Figure 5. Comparison of the survival curves of Escherichia coli and Micrococcus luteus on lter
papers under direct plasma exposure and remote plasma exposure (2 mm away from the plasma
glow). Argon atmospheric plasma conditions are 1500 sccm argon and 15 W dc power input.

A high-ow gas higher than 500 sccm is necessary in

order to blow the argon glow discharge out of the discharge
chamber and form a free brush-shaped plasma jet. To determine if the high owing plasma gas could blow the bacteria
cell off the supporting media, the blowing effect of argon gas
on the two bacteria was examined by owing the argon gas at
the same ow rate (1500 sccm) without igniting an electrical
discharge.
Morphological Examination

The effects of plasma treatment on the structures of the

bacteria were examined using a scanning electron microscopy
(SEM) (Model S4700, Hitachi, Tokyo, Japan). The bacteria
solutions of the controls and the plasma treated samples were
placed in the xation and then coated with a thin layer of
plasma sputtered platinum (Pt).
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RESULTS AND DISCUSSION

Effect of Study Parameters

For a given set of plasma conditions and medium, the sterilization was more effective on M. luteus compared to E. coli
(Figure 3). It was found (Figure 3) that a longer plasma
treatment time is required for killing E. coli than killing M.
luteus. Two reasons are advanced for this nding. The rst is
the difference in the cell structure of the bacteria. E. coli, a
gram-negative bacterium, has an outer membrane that restrains the direct attack of various plasma species, while M.
luteus (a gram-positive bacterium) does not have the outer
membrane in its cell structure.17 The second reason is that,
upon exposure to the plasma, a few E. coli bacteria survive,
which could start proliferating in the medium before the
bacteria count is commenced.18

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Figure 6. Sterilization effects of (A) dry heat at 125C and (B) gas blowing at 1500 sccm on
Escherichia coli and Micrococcus luteus seeded on lter paper and nutrient broth. [Color gure can be
viewed in the online issue, which is available at www.interscience.wiley.com.]

For a given set of plasma conditions and bacterium, the

sterilization is the fastest in the nutrient broth and the
slowest in the lter paper (Figure 3). This is because of the
direct attack of plasma species on the bacteria cells in the
nutrient broth medium. It was observed that the droplets of
the nutrient broth dried out within 1 min plasma exposure.
This enabled direct contact between the plasmas and the
bacteria cells. With the nutrient broth as the supporting
medium, no stain of E. coli and M. luteus was observed
after 4 min of plasma treatment. A possible reason for the
very different plasma sterilization time is the plasma penetrating efciency with different culture media. The lter
paper used in this study is a porous solid and requires
plasma penetration to kill the bacteria that resided inside
the pores. As a result, sterilization on the lter paper
required a longer plasma treatment time. The colloid state
of agar could slow down the plasma penetration but was

eventually destroyed through evaporation of water with

longer plasma treatment time.
For a given set of bacterium and supporting medium, the
efciency of sterilization increases with an increase in the
power input of the plasma source, and is affected by the argon
gas ow rate to a much less extent (Figure 4). In creation of
atmospheric plasmas, the power input and gas ow not only
affect the plasma density, but also the plasma temperature
(Figure 2). Higher power input could result in higher degree
of ionization of the gas and thus increase the density of
various plasma species, which are the reactive agents in
inactivating the bacterium cells. The nding with respect to
argon gas ow rate may be explained by the fact that a higher
ow rate could bring more reactive plasma species out of the
discharge chamber and increase the chance of collisions and
interactions between the plasma species and bacteria cells,
therefore, resulting in slightly better sterilization efciency.
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217

For a given set of plasma conditions, the position of the

plasma has a marginal effect on the sterilization of E. coli but
a marked one on M. luteus (Figure 5). In atmospheric plasmas, the plasma species could lose its energy or reactivity in
a very short time because of the much higher collision frequency among the plasma particles. The life time of the
reactive plasma species in atmospheric plasmas is much
shorter when compared to that in low-pressure plasmas.19 As
a result, most of the plasma species, except the argon metastables (long-life electronically excited argon atoms) could
lose their reactivity dramatically in a remote position (away
from the glow). This nding (Figure 5) suggests that argon
metastables in remote atmospheric plasmas are very effective
in inactivating M. leutus but not E. coli.
Proposed Sterilization Mechanism

It was found that the sterilization was, indeed, due entirely to

the plasma and not to either the dry heat or the argon gas
blowing by itself (Figure 6). A possible sterilization mechanism of argon atmospheric plasma is the continuous impact
on the membrane of bacterial cells by energetic plasma species such as argon ions, metastable particles, electronically
excited neutrals, and very likely UV radiation generated in
the source. The characteristic energies of argon neutrals (including argon metastables and electronically excited argon
atoms) and argon ions are of the order of 15 eV and 11 eV,
respectively, which are much higher than the bonding energy
of the organic molecules constituting microorganisms.20
Thus, even pure argon plasmas have the ability to destroy
various microorganisms. Mendis and coworkers21,22 suggested that charged particles might cause the rupture of the
outer membrane to the bacterial cells. Therefore, the plasma
exposure to the bacteria cells could eventually damage the
cell structure or the cell membrane, and, consequently, destroys the viability of the cells.
SEM examination of cell structure changes of the two
bacteria (Figures 7 and 8) showed the loss of lipid moieties in
the cells after plasma treatment, as evidenced by cell damage,
reduction of the cell size, and distortion of the dead cell. It
can be seen (Figure 7) that E. coli cells were severely damaged and only some cell debris remained with the argon
plasma sterilized samples. The SEM observation on M. luteus
(Figure 8) provided the similar information, i.e. the bacteria
cells were fragmented by argon plasma treatment. After the
plasma treatment, the size of M. luteus cells was signicantly
reduced and the shape of the dead cells was distorted.
Another possible mechanism is the penetration of the cells
of the bacteria by oxidation species produced by the interaction between the argon gas in the plasma and the surrounding
ambient air in the vicinity of the plasma source, suggesting
that sterilization efciency could be increased by increasing
the stream of these species.
Study Limitation and Future Work

In this study, some of the experimental tests were conducted

using lter paper only (Figures 4 8) but not using all three
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Figure 7. Scanning electron micrographs of (A) untreated Escherichia

coli control and (B) 5 min plasma treated Escherichia coli seeded on
lter paper under plasma condition of 1500 sccm and 15 W dc power
input.

support media. The major limitation of this study is that the

bacterial inactivation effects of the atmospheric plasma
source were investigated using only two kinds of bacteria, E.
coli and M. luteus with two measurements per test. In order
to fully determine the efciency of the plasma sterilization
method, all viable microorganisms, such as virus, fungi,
vegetative bacteria, and bacterial endospores, should be studied and large datasets should be acquired for performing
meaningful statistic analysis.
This study is our rst step to demonstrate the applicability
of the low-power atmospheric plasma source in a surface
sterilization/decontamination application. Investigations focusing on the elucidation of the plasma chemistry effects on
sterilization efciency are being performed in our laboratories and the results will be reported in due course. Future
work will include studies of the optimization of the plasma
conditions, statistical evaluation of the plasma sterilization
efcacy using a broad spectrum of microorganisms, including

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YU ET AL.

source as a promising alternative surface sterilization/decontamination technique.

The authors would thank the personnel of Electron Microscopy
Core Facility at MU for performing the SEM measurements.

REFERENCES

Figure 8. Scanning electron micrographs of (A) untreated Micrococcus luteus control and (B) 5 min plasma treated Micrococcus luteus
seeded on lter paper under plasma condition of 1500 sccm and 15
W dc power input.

those bacterial spores, virus, and fungi that are known to be

very difcult to kill.

CONCLUSIONS
This study demonstrated the rapid bacterial inactivation/sterilization capability of the low-temperature argon plasmas
created under one atmospheric pressure with a very low dc
power input. The bacterial inactivation effects of argon plasmas were conrmed and distinguished from the possible
synergetic effects of heat and fast gas blowing. The bacterial
inactivation/sterilization capability of the argon atmospheric
plasmas was mainly ascribed to the high-energy argon ions
and the electronically excited argon neutrals presented in the
plasmas. The plasma bacterial inactivation/sterilization capability demonstrated through this study indicated the tremendous potential of this low-power driven atmospheric plasma

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