Disinfection of Rhodococcus equi by
Atmospheric Pressure He/N2 Plasma Needle
Shahriar Mirpoura, Mahmood Ghorannevissa, Amir Hossein Saria ,Davoud Dorraniana
a. Plasma Physics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran
Abstract:
Rhodococcus equi is a facultative intracellular bacterial pathogen that can cause substantial morbidity in
patients that are immunocompromised and are occupationally and recreationally exposed to farming,
livestock, and dry soil environments. The objective of this study was to determine the in vitro
antimicrobial activity by means of non-thermal plasma (plasma needle). Rhodococcus equi was cultured
by plating the specimen onto nutrient agar medium; 2 samples from cultured plate were taken. In the first
sample bacterial colonies were transferred from the surface of the plate into 10 ml sterile saline (1
dilution) then for making the second one, 0.1 ml of this sample was transferred to a 9.9 ml sterile saline
blank (1/100 or 10-2 dilution). One ml of each samples were directly exposed to the plasma. The time and
distance between the plasma needle and the samples have been varied. As well two similar non-exposed
samples by plasma were considered as the control. After plasma treatment, the samples were incubated at
37°C for 48 -72 hours. To evaluate the effects of treatment on cellular count, the samples were examined
immediately after incubation. This paper showed that approximately half colony units were destroyed.
Prolongation of treatment significantly improves the destruction efficiency and details of results will be
discussed in the Rhodococcus equi could be efficiently deactivated using non-thermal plasma (plasma
needle).
Keywords: Atmospheric pressure glow discharges (APGD); Cold plasma; Plasma Needle
1. Introduction
Nowadays plasma has become increasingly
prominent in industrial applications like textile
treatments, material coating, etc. During the last
decade plasma has been introduced some
properties which demonstrate its usefulness and
promising utilization in many biomedical
applications [1]. The most
important
characteristic of non- thermal atmospheric
pressure plasma (NTAPP) is that the gas
temperature is close to room temperature so the
thermal damages are considerably low. Free
radicals, chemical reactive species, ion,
metastable and energetic particles, molecules,
and UV photon play an important role in
disinfection process by NTAPP [2-4].
connected to a RF power supply antenna (13.56
MHz, Yarnikan Saleh Co). The RF power
measurement (the forwarded and reflected
power) was provided by a RF matching box. The
forwarded power was 23 Watt for all of the tests
in the study.
The working gas was pure helium (with purity
percentage of 99.999) The schematic view of the
plasma needle and its real photo during a typical
experiment are demonstrated in figures 1 (a) and
(b), respectively.
Using common sterilization method such as
autoclaves, chemicals like EtO (Ethylen Oxide)
and irradiation of gamma ray are associated with
some risks for both operators and environment.
Limitations of the methods including thermal
damage, risk of toxicity of chemicals, and also
destructive effects of radiations encourage
investigators to try and replace alternative
methods [3,5-6]
Rhodococcus equi is a facultative intracellular
bacterial pathogen which can cause substantial
morbidity in patients who are immunecompromised and are occupationally and
recreationally exposed to farming, livestock, and
dry soil environments [7-9].
Many diagnostic tests became available for
evaluation of the cell changes during plasma
treatment including SEM analysis, absorbance
spectrum for protein leakage [3].
2. Experiments and Methods
2.1. The plasma needle setup
The plasma needle consists of a thin tungsten
wire with sharp tip (diameter = 0.3 mm) as the
central electrode insulated with a Pyrex tube
(inner diameter = 1mm) which confined with
another Pyrex tube as the nozzle (inner diameter
of 4.5 mm and length of 3 cm). The length of
under exposure part of the metal wire of the
glass insulator was 5 mm. The electrode was
Fig1. Plasma needle device (a) schematic view, (b) real photo in a
typical experiment
The flow rate of filling gas was 1 lit/min which
controlled by a flow-meter. This rate remained
constant during all of the experiments.
Moreover, 2% of pure Nitrogen (N2) was
injected as addition gas into some experiments.
In order to investigate the effect of exposure
time and distance between plasma plume and
sample, two series of tests were performed for
both He and He/N2 plasma. In first series of
experiments, in a constant exposure time (60 s)
the distance between the electrode tip and Petri
dishes changed from 1 mm to 3 mm. In the
second series of experiments, treatment time
varied from 10 to 180 s in a fixed 2 mm distance
between the needle’s tip and sample.
The temperature of the plasma in afterglow
region (about 2 mm) was measured by a
thermocouple during the experiments. The
maximum temperature of the He plasma in the
discharge zone, afterglow region, and the remote
area, for 3 min treatment period were 48ºC,
32ºC, and 26ºC respectively. These temperatures
reached
to
57ºC,
38º
C, 30ºC for He/N2 plasma treatment.
2.2. Bacterial Colonial Culture
The Gram-positive Rhodococcus equi ATCC
6939 was cultured by plating the specimen into
nutrient agar medium. Bacterial colonies were
transferred from the surface of the plate into 10
ml sterile saline for making bacterial suspension.
This suspension level was compared to the 0.5
McFarland standards. Then in order to make a
sample of 0.1 ml dilution, bacterial suspension
was transferred to a 9.9 ml sterile saline blank
(1/100 or 10-2 dilution). One ml of sample was
transferred to Petri dish and then was directly
exposed to the plasma plume.. Furthermore, a
similar unexposed sample was considered as the
control. After plasma treatment with various
experimental
conditions,
the
melted agar/bacterial suspension was mixed and
poured evenly across the top of an agar plate and
was allowed to solidify then the samples were
incubated at 37°C for 48 -72 hours. To evaluate
the effects of treatment on cellular count, the
samples were examined immediately after
incubation and the number of CFU for
dilution 0.01 counted and calculated the number
of bacteria in the original suspension.
The CFU counting of treated samples performed
for both He and He/N2 plasma. Fig.2 shows the
germicidal effect at different exposure time for
bacteria dilution of 0.01. It can be observed from
the figure that by increasing the plasma
treatment time the germicidal effect enhance. In
addition, we realized that in pure He plasma
treatment, even after 180 s sterilization did not
complete while in He/N2 plasma after 120 s
there was not any colony forming in Petri dishes.
All the tests for time variations experiments
performed in afterglow region.
Fig2. Germicidal effect in different variable time
3. Results
3.1 Germicidal Effect
After bacteria’s incubation, the germicidal effect
factor (GE) was drawn for both time and
distance variations. The formula of the GE is
determined as eq.1:
GE = Log (Ni /Nf)
(1)
Where Ni and Nf are the number of the CFU
(Colony Forming Unit) in control (1.5*108) and
plasma treated plates, respectively [24].
Fig3. Germicidal effect at different distance between plasma and
samples.
Fig.3 shows the germicidal effect when the
distance between plasma plume and sample
varied from 1 to 3mm. After 3 mm we reached
to the remote area of plasma. The results show
that by decreasing the distance from 3 to 1 mm,
germicidal effect increases. This could be caused
by approaching to electrode tip and transition
from afterglow region to discharge zone. It
should be considered that in discharge zone
plasma temperature and density is higher than
afterglow region and remote area [10].
Meanwhile, the distance between the electrode
and sample may have influence on the
deactivation of bacterial colonies. Based on this
fact, our results demonstrated the germicidal
effect of three plasma zones including: Remote
area, Afterglow region, Discharge zone was
enhanced, respectively. Transition zone plasma
may be resulted in the increase the temperature
and ionized species density. The results of this
experiment were compactable with the results of
Yang et al [10].
4. Discussion
5. Conclusion
In the present study we proved that cold plasma
could be utilized to inactivate Rhodococcus
equi. Sladek et al. stated that E. coli colonies can
be efficiently deactivated using NTAPP by
helium–air mixtures [2]. They proved that the
corresponding numbers of destroyed CFUs rise
with treatment time, but it is saturated
approximately after 50 s. Further increase of the
treatment time to more than 60s did not improve
the deactivation process. In our study prolonged
plasma treatment had distinguishable effect on
deactivating of bacterial colonies. We obtained
complete decontamination after 120 s. This
difference between our study and Sladek
investigation could be attributed to the
significantly different effect of more forwarded
power and longer distance between electrode
and bacterial sample. Moreover, it seems that
using different bacterial samples (E.Coli vs
Rhodococcus equi) may have a large effect on
the achieved results [11].
This study demonstrates the germicidal effect of
atmospheric pressure He/N2 plasma needle on
Rhodococcus bacteria. Based on the findings of
the present study it can be concluded that by
adding nitrogen gas to the helium plasma the
germicidal effect enhanced. In addition, by
increasing the plasma treatment time the
germicidal effect was enhanced.
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Acknowledgment
The work was supported by Science and
Research Branch of Islamic Azad University.
We are indebted to Dr. Dorranian, Ms. N.J.
Farahani and Mr. M.H. Afzali for their valuable
consultations
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