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IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 37, NO. 12, DECEMBER 2009 2347

Inactivation Characteristics of Bacteria inCapacitively Coupled Argon Plasma

Sureshkumar and Sudarsan Neogi

AbstractPlasma technology is being focused on the medical,food, and pharmaceutical fields for sterilization applications. Thesterilizing effect of the 13.56-MHz radio-frequency (RF) plasmagenerated by using argon gas was studied using Staphylococcusaureus, one of the most common pathogens liable to hospital-acquired infections. The major focus of this paper was to performa parametric study by varying the external-process parameterssuch as plasma treatment time, RF power, gas-flow rate, and pres-sure on the inactivation ofS. aureus. The results were supported byoptical emission spectroscopy and scanning electron microscopystudies.

Index TermsBiomedical engineering, plasma applications,sterilization, surface contamination, surface treatment.

I. INTRODUCTION

THE staphylococcus aureus is one of the most important

pathogens responsible for hospital-acquired infections.

Any sterilization process must ensure the complete removal of

microorganisms relevant to the targeted application and must

leave the material with no or minimal damage. Sterilization is

the complete elimination of all types of microorganisms [1].

Sterilization can be achieved by heat, chemicals like ethylene

oxide, radiation, pulsed light, and plasma. Each sterilization

method has its own limitations. For instance, heat (either dryheat or steam) sterilization cannot be used to sterilize heat-

sensitive materials since deformation of such materials may be

expected at high temperatures. Chemicals like ethylene oxide

can sterilize materials at low temperatures. However, the prod-

ucts sterilized by EtO sterilization should be aerated for a longer

time before use [2]. Since EtO is flammable and carcinogenic,

adequate safety measures are needed. Radiation sterilization is

commercially used nowadays for sterilizing single-use devices.

It needs an isolated site of operation, and proper safety mea-

sures should be taken while working with gamma radiation. It

has been reported that certain polymers like PTFE, polyacetals,

etc., are not compatible with gamma radiation [3]. Plasma

sterilization has been in focus in recent years because of itsremarkable advantage of ambient-temperature operation, high

efficiency, shorter treatment times, and nontoxicity. Contrary

to EtO, sterilization by using plasma is considered to be safe,

both for the operator and the patient [1]. Sterilization by plasma

Manuscript received August 4, 2009; revised September 15, 2009. Currentversion published December 11, 2009. This work was supported by theDepartment of Science and Technology, India (DST Grant SR/S3/CE/31/2005).

The authors are with the Department of Chemical Engineering, IndianInstitute of Technology Kharagpur, Kharagpur 721 302, India (e-mail: sneogi@che.iitkgp.ernet.in).

Digital Object Identifier 10.1109/TPS.2009.2033112

can be achieved at atmospheric pressure or in vacuum. At the

present condition, the sterilization technology is still in an iso-

lated and scattered experimental stage [4]. There are also other

sterilization methods which use high electric field [5], high-

voltage pulse [6], and pulsed-light irradiation [7]. However, in

the aforementioned methods, complete sterilization is achieved

only at higher temperature. Plasma is a partially ionized, low-

pressure gas that is composed of ions, electrons, and UV

photons as well as the reactive neutral species (free radicals

and excited atoms) with sufficient energy to break covalent

bonds and initiate various chemical reactions [2]. Low-pressureplasmas are generated in the pressure range of 1100 Pa

(133 Pa = 1 torr) [8]. For any application in plasma, it isnecessary to have a basic understanding of the experimental

system used by playing with various process parameters.

In the current investigation, the sterilizing effect was studied

by varying the plasma parameters using a model organism

(S. aureus) in a low-pressure plasma reactor. The effects of

treatment time, radio-frequency (RF) power, gas-flow rate, and

gas pressure were studied using argon plasma. Similar para-

metric studies were reported using different configurations of

plasma systems such as Escherichia Coli with remote glow-

discharge air plasma [9], Bacillus subtilis with low-pressureoxygen-based plasmas in distributed electron cyclotron reso-

nance reactor [10] and Escherichia Coli with remote argon

plasma [11].

II. EXPERIMENTAL SETUP

A. Plasma Reactor

A capacitively coupled plasma reactor was used for this

study, as shown in Fig. 1. The stainless-steel reactor consists

of two parallel plates fixed inside a cylindrical chamber. The

reactor was connected to an RF generator, which generates

plasma in the RF range of 13.56 MHz, through a matchingnetwork. Gas-flow rate was controlled by means of a mass-flow

controller. The temperature inside the sterilization chamber was

measured by means of a thermocouple, and gas pressure was

monitored by means of a capacitance gauge.

The external parameters such as gas pressure, gas-flow rate,

and time of exposure to plasma for each experimental run

can be prefixed using control systems. Initially, the reactor

was pumped down to a pressure of about 0.76 mtorr using

a Roots and rotary-pump cascaded arrangement. Then, argon

gas was allowed to enter the chamber through a mass-flow

controller. Once the pressure inside the sterilization chamber

was stabilized, the RF plasma was ignited.

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2348 IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 37, NO. 12, DECEMBER 2009

Fig. 1. Reactor setup used for sterilization study.

TABLE IOPERATING CONDITIONS OF THE PLASMA REACTOR

B. Plasma Treatment

The S. aureus (NCIM 2079) chosen for the sterilization study

was procured from NCIM, Pune, India. The bacterial strain

was maintained on standard-method agar slants at 56 C. The

culture was grown on a sterile nutrient broth, containing 5 g/l of

peptic digest of animal tissue, 5 g/l of sodium chloride, 1.5 g/lof beef extract, and 1.5 g/l of yeast extract incubated in a

rotary shaker operating at 175 r/min and maintained at 37 C.

For the sterilization studies, the cells were harvested from the

exponential phase for plasma treatment. A drop volume of 50 l

of the bacterial suspension was aseptically applied over the

sterile glass slides of dimension 25 mm 25 mm 1 mm

and left in the laminar hood for drying. The samples (slides

containing dried bacteria) were placed inside the sterilization

chamber for plasma treatment. Purple, the characteristic color

of argon, was observed inside the reactor. After plasma treat-

ment, the chamber was vented to the atmosphere, and the

samples were collected. The bacterial samples were treatedfor different parameters as mentioned in Table I. One of the

samples was not exposed to plasma and kept as a control

sample. The treated samples were collected and serially diluted

using sterile 0.02 M phosphate buffer at pH 7. A drop volume

of 50 l of the diluted suspension from each test tube was

plated on the sterile nutrient agar plates using standard agar

spread-plate procedure. The plates were observed for bacterial

growth after incubation at 37 C for 24 h. After incubation,

S. aureus colonies were observed over the surface of the agar

plates, which were confirmed by gram staining.

The number of bacterial colonies formed over the agar plates

was counted visually. A semilogarithmic graph was plotted

between the number of colonies remaining after treatment andthe plasma treatment time, which is called the survivor curve, to

determine the D-value. D-value is defined as the time required

to reduce a bacterial population by 90%. D-value is obtained

from the inverse of the slope of the survivor curve [12].

To determine the effect of individual plasma parameters,

germicidal effect (GE) was calculated. GE is another way of

expressing or validating the efficiency of a sterilizing process.GE is defined as the difference between the logarithm of the

original bacterial population (No) and number of survivors(Nt) remaining after plasma treatment. It is expressed as GE =logNo logNt [13].

C. OES

Optical Emission Spectroscopy (OES) is a noninvasive

technique for the diagnosis of active species present in the

plasma. The light emitted by plasma was captured by means

of a high-resolution optical emission spectrometer (HR4000

CG-UV-NIR) from Ocean Optics Inc., U.S. The spectral res-

olution was 1 nm, and the hit interval was 0.1 nm.

D. SEM

Scanning electron microscopy (SEM) studies were car-

ried out in a scanning electron microscope (JEOL-JSM-5800,

Japan) to study the surface morphology of bacteria before and

after plasma treatment. Prior to analysis, the samples were

coated with a thin layer of gold.

III. RESULTS AND DISCUSSION

A. Effect of Plasma Treatment Time

The results for the effect of treatment time are shown in

Fig. 2. It is observed that by increasing the exposure time to

plasma, the number of colonies reduced. However, since the

only candidate for sterilization is argon plasma, the bacterial

reduction was less. This shows the effect of argon plasma on

S. aureus with respect to treatment time. The applied power

was maintained at 100 W for all treatment times. The results for

the bacteria reduction are plotted in a survivor curve as shown

in Fig. 2(a). A two-phase nature of the survivor curve was ob-

served. The first phase (D1 = 0.53 min) is due to the killing ofbacteria at the top surface of the bacterial load by UV radiation

from the argon plasma. This phase has a smaller D-value, as

already reported. It is known that UV rays in the wavelength of220280 nm are readily absorbed by DNA and, thereby, affects

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SURESHKUMAR AND NEOGI: INACTIVATION CHARACTERISTICS OF BACTERIA IN CAPACITIVELY COUPLED ARGON PLASMA 2349

Fig. 2. Effect of plasma treatment time (100 W, 40 sccm, 150 mtorr).(a) Survivor curve. (b) GE versus treatment time. (c) Comparison betweenexperimental and calculated data.

the replication or reproduction of the cell. On the other hand, the

second phase (D2 = 3.61 min) is due to the effect of the UVradiation and argon free radicals over stacked species, which

was revealed by a larger D-value. However, if the cells are

stacked, the cells or cell debris (formed as a result of Phase I)

lying over the top surface hinders the cells at the subsurface

from UV exposure [1]. Hence, a marginal bacterial reduction

was achieved. The GE was also plotted as a function of plasmatreatment time as shown in Fig. 2(b). Using the survivor curve,

Fig. 3. Effect of input RF power (5 min, 40 sccm, 150 mtorr).

a quantitative relationship was developed between the number

of survivors (CFUs/ml) remaining after plasma treatment and

plasma treatment time (t) which is represented by the followingpolynomial equation:

log(CFUs/ml) = 0.019 t4 0.255 t3

+ 1.261 t2 2.908 t + 8.66. (1)

The plot for (1) along with the experimental data is shown in

Fig. 2(c).

B. Effect of RF Power

The effect of input RF power on the sterilization efficiency is

shown in Fig. 3. The total energy input to the gas is increased byincreasing the input power. Hence, it is obvious that increasing

the input RF power increases the electron density by increasing

the number of active species in the plasma [14]. With pure argon

plasma, a significant bacterial reduction can be achieved by

increasing the power from 20 to 100 W. This shows that the

GE strongly depends on input RF power. At 20 W, the GE was

1.69, whereas it exceeded 3.0 while applying the input power

of 100 W for the same operating conditions. However, while

operating at higher power, the effect of temperature should

be also accounted. In this case, the temperature at the end of

treatment was observed to be 40 C.

C. Effect of Gas-Flow Rate on Sterilization

As can be observed from Fig. 4, the GE varied between

1.5 and 3.1 while varying the argon gas-flow rate from 20 to

100 sccm (standard cubic centimeters per minute). At higher

flow rates, the bacterial reduction (GE) was much weaker than

at lower flow rates. This effect has been studied by several re-

searchers [9], [10]. Increasing the flow rate reduces the average

residence time of molecules inside the sterilization chamber.

At a given constant power, only a given or possible number

of molecules gets ionized and results in plasma formation. In

the current experimental setup, a significant difference was ob-

served by varying the argon-gas-flow rate from 20 to 100 sccm.It can be concluded that the lower flow rates showed relatively

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2350 IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 37, NO. 12, DECEMBER 2009

Fig. 4. Effect of gas-flow rate (100 W, 5 min, 150 mtorr).

better bacterial reduction than the higher flow rates [15]. Ifthe gas-flow rate is too low (as in the case of 20 sccm), the

number of reactive species generated might be less enough

for efficient sterilization as reflected from lower GE values.

Hence, an optimum flow rate should be selected for efficient

sterilization. The sterilizing effect improves until a saturation

point is reached for the applied power, i.e., the maximum

number of argon atoms is getting ionized at this point for an

applied power of 100 W and, thereby, resulted in a higher GE

value is at 40 sccm. However, the average energy of the species

in the plasma and the residence time is lower at higher flow

rates than at low flow rates [11]. This might be the reason for

greater GE at low flow rates, and after reaching a peak value

at a particular flow rate, it reduces. In the present experimentalconditions, better sterilization was achieved when the gas-flow

rate was maintained at 40 sccm.

D. Effect of Gas Pressure

It is known that an increase in pressure reduces the gas mean

free path. As the atoms get more crowded inside the chamber,

the collision rate increased, and there is a slight increase in

plasma density, i.e., the number of active species per unit

volume. In this experimental setup, the working pressure for

RF plasma generation can be varied from 100 to 200 mtorr. It

is obvious from Fig. 5 that there is no significant difference inbacterial reduction with the variation of pressure in the given

range. Since, at higher pressures, the mean free path of the

gas and electrons reduced; collisions among them increase to

generate active species in plasma. However, the average energy

of the active species becomes less due to frequent collisions

inside the sterilization chamber, which in turn, decreases the

sterilization efficiency [16]. This effect resulted in relatively

lower GE value at 200 mtorr.

E. OES

To realize the species present in argon plasma, OES studieswere performed. The optical emission spectrum generated by

Fig. 5. Effect of gas pressure (100 W, 5 min, 40 sccm).

argon plasma is shown in Fig. 6. It is understood from Fig. 6

that the neutral and atomic species of argon were present in

the plasma. The presence of UV radiation, which is consid-

ered to be the dominant species for pure argon plasma, was

also observed in the range of 308.10 nm. OES studies were

discussed with each variable plasma parameter as given next.

Neutral argon emission lines were observed at 601.75, 800.62,

912.30, 922.45, and 965.78 nm. The presence of argon ions

were indicated by peaks observed at 686.13, 771.72, 794.05,

841.57, and 922.76 nm.

1) Effect of Treatment Time: By increasing the plasma treat-

ment time, a marginal change in the plasma spectrum wasobserved. As mentioned earlier, an emission band was observed

near the 300310-nm range indicating the presence of UV

radiation. Similarly, the respective peaks for argon atoms and

ions (both single and multiple) were also observed.

2) Effect of Power: Increasing the input power increased

the number of active species present in plasma due to the

increase in energy input. This effect was well realized by OES

studies. For instance, the intensities of the argon neutral (Ar I

601.75) atoms and ion (Ar II 736.54 nm) peaks were found to

be increasing during an increase in the input RF power. This

showed the presence of large concentration of plasma species

at higher powers.3) Effect of Gas-Flow Rate: Since the flow rate of argon is

increased, there is a decrease in plasma density by flushing out

the plasma. Fig. 7(b) shows the slight increase in the intensity

of the argon neutral (601.75 nm) and ion (811.09 nm) peaks.

Since the power and the gas pressure are fixed for the constant

reactor volume, an optimum number of atoms are ionized

within the given treatment time at a given flow rate for bacterial

sterilization.

4) Effect of Pressure: Increasing the gas pressure inside the

sterilization chamber reduces the mean free path of electrons.

This, in turn, increases the number of collisions between atoms

and electrons; however, the average energy of these plasma

species gets reduced. A decrease in the intensity of the peakswas observed as the gas pressure is increased, as shown in

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SURESHKUMAR AND NEOGI: INACTIVATION CHARACTERISTICS OF BACTERIA IN CAPACITIVELY COUPLED ARGON PLASMA 2351

Fig. 6. Optical emission spectrograph of argon plasma.

Fig. 7. (a) Argon species intensity variation with power. (b) Argon speciesintensity variation with gas-flow rate. (c) Argon species intensity variation withgas pressure.

Fig. 7(c). For instance, by increasing the pressure from 100 to

200 mtorr, the intensity of the neutral argon peak at 601.75 nmreduced from 1962 to 1535 arbitrary units.

Fig. 8. SEM images. (a) Control S. aureus cells. (b) After 5 of min argonplasma treatment.

F. SEM

SEM studies were carried out for control and treated bacterial

cells. The images captured by scanning electron microscopeare shown in Fig. 8(a) and (b). The treated cells shown here

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