1 s2.0 S0257897210000861 Main
1 s2.0 S0257897210000861 Main
1 s2.0 S0257897210000861 Main
a r t i c l e i n f o a b s t r a c t
Available online 6 February 2010 Cold atmospheric plasma treatment of microorganisms and living tissues has become a popular topic in
modern plasma physics and in medical science. The plasma is capable of bacterial inactivation and non-
Keywords: inflammatory tissue modification, which makes it an attractive tool for treatment of skin diseases, open
Atmospheric plasma injuries and dental caries. Because of their enhanced plasma chemistry, Dielectric Barrier Discharges (DBDs)
Dielectric barrier discharge have been widely investigated for some emerging applications such as biological and chemical
Plasma sterilization
decontamination of media at ambient conditions. Despite the high breakdown voltage in air at atmospheric
E. coli
S. aureus
pressure, the average current of DBD discharges is low. Therefore, a DBD can be applied in direct contact with
biological objects without causing any damage. In this work a 60 Hz DBD reactor, which generates cold
atmospheric plasma inside Petri dishes with bacterial culture, is investigated. Samples of Staphylococcus
aureus, a Gram-positive bacterium and Escherichia coli a Gram-negative bacterium were selected for this
study. The bacterial suspensions were evenly spread on agar media planted in Petri dishes. The reactor
electrodes were placed outside the Petri dish, thus eliminating the risk of samples microbial contamination.
The covered Petri dish with agar medium in it serves as dielectric barrier during the treatment. The plasma
processing was conducted at same discharge power (∼ 1.0 W) with different exposure time. Sterilization of
E. coli and S. aureus was achieved for less than 20 min. Plasma induced structural damages of bacteria were
investigated by Scanning Electron Microscopy.
© 2010 Elsevier B.V. All rights reserved.
0257-8972/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.surfcoat.2010.01.052
K.G. Kostov et al. / Surface & Coatings Technology 204 (2010) 2954–2959 2955
However its role in air plasma sterilization at atmospheric pressure is Escherichia coli a Gram-negative bacterium were selected for this
considered to be modest [11], since no significant UV emission occurs study. Closed Petri dishes, planted with agar media containing E. coli
below 285 nm. In high-pressure non-equilibrium plasma discharges, or S. aureus, were placed between two metal electrodes connected to a
reactive species are generated through various collisional ways, such high-voltage transformer. All plasma treatments were conducted at
as electron impact excitation and dissociation [11]. The active free same discharge power (∼1.0 W) with different exposure time. All
radicals abundant in DBD plasma, particularly the reactive oxygen bacterial cells were killed for less than 20 min of DBD treatment.
species (O, OH, O− 2 , O3) are considered to be the prime atmospheric Plasma induced structural damages of bacteria were investigated by
plasma disinfectants [11–13]. Finally, the exact role of charged Scanning Electron Microscopy (SEM).
particles in bacterial inactivation is not yet fully resolved, but there
are some indications of their importance [2,9]. At atmospheric
pressure DBD discharges the average energy of the ions is quite low 2. Experimental procedure
(on the order of 10−2 eV). Therefore the cell physical damage induced
by bombardment with low energy charged particles is expected to The experimental arrangement used for DBD bacterial sterilization
play no direct role in the destruction of microorganisms. In analogy to is shown in Fig. 1. Low-temperature plasmas at atmospheric pressure
the charging of dust in complex plasmas one can consider a negative were generated inside covered Petri dishes (90 × 15 mm, Inlab, São
charging of the bacterial cells during the plasma exposure. The Paulo, Brazil) that were partially filled with 20 ml culture medium
authors [2] suggest that the mechanical stress induced by electrostatic (Triptic soy agar, Oxoid, Hampshire, England) leaving air spacing of
repulsion forces of the charged surface may under certain conditions about 5 mm.
cause cell rupture in Gram-negative bacteria because they possess fine Reference strains E. coli ATCC 23922 and S. aureus ATCC 6538 were
membrane and irregular shape. Some researchers [14] reported cells used in this study. These two species are commonly employed as the
morphological changes after direct plasma exposure, which were reference microorganisms in the development of new sterilization
attributed to the charge accumulation on bacteria. Furthermore, techniques, representing Gram-negative and Gram-positive bacteria,
bacteria are very sensitive to changes in their surface charges. respectively. Suspensions of S. aureus or E. coli were standardized to
Therefore disturbing the surface charge equilibrium of bacteria by concentration of 1 × 104 cells/ml by optical spectrophotomety (the
charged particle bombardment may actually kill the cell without its values of respective wavelengths and optical densities were: 490 nm
lysis, what probably happen with more robust Gram-positive bacteria and 0.366 for S. aureus and 590 nm and 0.319 for E. coli). Portions of
[2,14]. 0.1 ml of the initial suspensions were evenly spread on the surface of
The nature of bacteria inactivation by cold atmospheric plasma is the agar medium, then left to dry for 15 min under controlled
rather complex and the exact mechanisms of cellular death are still conditions inside a laminar flow chamber and, finally treated by
under discussion [3]. However, the kinetics of the bacterial inactiva- plasma. The discharge was driven by a low-frequency high voltage
tion can provide some important information for the process. A simple power supply operating at 60 Hz. Two 75-mm-diam, aluminum
and reliable approach is based on the dynamical studies of bacterial electrodes were attached outside the Petri dish, thus preventing
growth after plasma treatment, that involve planting and counting the microbial contaminations of the samples. The high voltage electrode
number of colony forming units (CFU). Typically, dose–response (or was placed on the Petri dish's cover and the electrode under the Petri
survivors) curves are determined, i.e., the numbers of CFUs are dish was grounded.
measured in function of the plasma exposure time. The most common Applied voltage was measured by a high-voltage probe Tektronix
forms of the survivor's curves for air plasma treatment are TDS 2024B. The current waveforms were recorded by measuring the
exponentially decaying functions with a single or multiple time voltage across a 1200 Ω resistor connected in series with the reactor.
constants [2,3]. These curves are usually plotted in semi-logarithmic The discharge current and the applied voltage are shown in Fig. 2. To
scales and the time for a one-log10 reduction is referred to as D-value obtain the charge flowing through the gap the resistor was
(Decimal value) — the time required to reduce an original concen- substituted by a 0.91 μF capacitor. From the Q–V Lissajous figure
tration of microorganisms by 90%. The D-values of air plasma (see Fig. 3) the discharge energy per cycle is calculated using the
deactivation can range from couple of seconds up to several minutes standard method based on the figure's area. The mean power is
depending on the type of microorganisms, the kind of medium obtained by multiplying the energy by the source frequency.
supporting the microorganisms, the method of plasma exposure During all experiments the magnitude of the applied voltage was
(remote or direct) and the treatment parameters [2,11,14]. fixed at 20 kV, which corresponded to mean discharge energy per
In this work we describe a 60 Hz DBD reactor, which generates cycle of about 17 mJ (electric power of 1.0 W). To examine the DBD
cold atmospheric plasma inside a Petri dish with bacterial culture. sterilization efficiency the plasma exposure time was varied from
Samples of Staphylococcus aureus, a Gram-positive bacterium and 1 min to 20 min.
Fig. 4. (A) Images of Petri dishes showing E. coli colonies after air plasma treatment for 0; 2; 5; 10; 15 and 20 min and (B) the E. coli survivor curve.
enough to produce some structural damage on the cells membrane the cells and the plates. The fixation process included immersion in
upon hitting a bacterium. Besides the bombardment with charged 2.5% glutaraldehyde for 1 h. Afterwards the slides were dehydrated by
particles, the accumulation of electric charge on the cell membrane submission to ethylic alcohol solutions with sequential concentra-
induces electrostatic stress, which can eventually rupture cells [2]. tions of (25%, 20 min; 50%, 20 min; 75%, 20 min; 90%, 20 min; and
The air plasmas are excellent sources of reactive oxygen and 100%, 60 min).
nitrogen species such as atomic oxygen, ozone, hydroxyl group, NO, As processed glass plates containing S. aureus and E. coli cells were
NO2 etc. These highly reactive species have direct impact on the coated with an ultra-thin layer of gold and micrographs were
microorganisms, and especially on their outmost membranes by subsequently taken for morphological inspection. SEM images of the
chemical etching [3]. Although, the exact sterilization mechanisms treated and control samples are shown in Figs. 6 and 7.
using plasma is still unclear most researchers have concluded that the As can be seen in Fig. 6, the Gram-positive bacteria S. aureus
simultaneous action of both processes — the chemical etching suffered structural damage when exposed to the DBD plasma. Many
performed by reactive neutral species as well as the physical damages cells have their outer membrane disrupted leading to release of their
of the cells membrane due to the ion bombardment and the charge cytoplasm. The Gram-positive bacteria have thicker membranes
accumulation, are important for the cells inactivation [2,6,13]. providing them with higher tensile stress and rigidity in comparison
To investigate the effects of plasma exposure on the bacterial to the G-negative bacteria. Probably, that is why some cells in Fig. 6(B)
structure Scanning Electron Microscopy (SEM) was used to inspect exhibit no visible morphological damages in their structure although
E. coli and S. aureus morphology before and after the DBD treatment. they may have lost their vitality.
Suspensions of E. coli and S. aureus were inoculated on sterile glass Gram-negative bacteria have thinner outer membrane and
slides (5 × 5 mm), which were put inside a covered Petri dish to form a therefore they are more susceptible to the attack of charged particles
DBD reactor similar to that used in the previous experiment. The and active radicals abundant in plasmas. Apparently the cells of
samples were treated for 15 min at applied voltage of 20 kV. Prior the plasma treated Gram-negative bacteria E. coli in Fig. 7(B) do not
SEM analysis plasma treated samples and untreated controls were represent apparent cytoplasm leakage. On the other hand, some cell
subjected to the following procedures to develop adhesion between fragments can be seen and the membranes of all bacteria in Fig. 7(B)
2958 K.G. Kostov et al. / Surface & Coatings Technology 204 (2010) 2954–2959
Fig. 5. (A) Images of Petri dishes showing the viable S. aureus colonies after plasma exposure for 0; 2; 5; 10; 15 and 20 min and (B) the S. aureus survivor curve.
look somehow eroded when compared to the cells membrane on the The electric power applied in this experiment was quite low
control sample (Fig. 7(A)). Based on the SEM micrographs we can (∼ 1.0 W) therefore longer treatment times were required to
infer that the DBD treatment induces some structural changes in the inactivate the bacterial strains. One may speculate that such a plasma
bacterial surface leading to lysis of the S. aureus cells and to the treatment in which the mean energy (∼ 17 mJ) is delivered during
membrane erosion of E. coli. long time (tens of min.), does not necessarily compromise the entire
Fig. 6. SEM images of (A) untreated S. aureus and (B) S. aureus cells treated for 15 min.
K.G. Kostov et al. / Surface & Coatings Technology 204 (2010) 2954–2959 2959
Fig. 7. SEM images of (A) untreated E. coli and (B) E. coli treated for 15 min.
structure of bacteria. That is probably why the SEM micrographs species and the physical damage of the bacteria outer membranes
demonstrate that some S. aureus cells look intact and the membranes induced by charged particles are involved in the sterilization process.
of E. coli seem eroded but not fully disrupted. Despite of this, both
bacterial strains were inactivated for about the same time of 20 min. References
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