Fluorescent observation of Penicillium digitatum on atmospheric pressure plasma treatment
Takayuki Ohta1, Takumi Mori1, Masafumi Ito2, Masaru Hori3
1
2
Faculty of Systems Engineering, Wakayama University, 930, Sakaedani, Wakayama 640-8510, Japan
Faculty of Science and Technology, Meijo University,1-501, Shiogamaguchi, Tenpaku-ku, Nagoya 468-8502, Japan
3
Department of Electrical Engineering and Computer Science, Nagoya University,
Furo-cho, chikusa-ku, Nagoya 464-8603, Japan
Abstract: The effect of plasma treatment on cell membrane of the spores of Penicillium digitatum
was observed by using fluorescent microscopy with the diI (1,1'-Dioctadecyl-3,3,Y,3'-tetramethylindocarboeyanine perchlorate) which stained the cell membrane in order to investigate the effect of
reactive oxygen species. The spores treated by the non-equilibrium atmospheric pressure plasma
were compared with that by the ultraviolet treatments. The function of cell membrane was inhibited
by the plasma treatment and the organelle was stained in the case of plasma treatment.
Keywords: non-equilibrium atmospheric pressure plasma, inactivation, Penicillium digitatum
1
Introduction
Inactivation of microorganisms using a plasma processing method has attracted much attention recently,
especially as a substitute for medical instrument sterilization methods. A plasma processing method possesses
many advantages such as a low-temperature treatment
and short processing time. Plasma inactivation shows
promise as a very effective system, which causes minimal damage to the instruments.
Inactivation factors, such as ultraviolet-C (UV-C) emission between 100 and 280 nm, neutral species, charged
species, electric field, and synergic effects, for various
plasma processes from low pressure to atmospheric
pressure have been intensively studied.[1] Investigation
of the inactivation factor based on the quantitative
diagnostics on the gas phase and the change of cell after
the plasma treatment is required. We have reported that
the rapid inactivation of spores of Penicillium digitatum
using high density nonequiribrium atmospheric pressure
plasma (NEAPP) [2]. The green mold of citrus is caused
by spores of Penicillium digitatum. Spores of P. digitatum which is fungus are different from other microorganisms for medical applications because of the resistant
structure, composition, and function of their cell wall. We
investigated the inactivation effects of ozone and UV
radiation. These results showed that the inactivation rate
of this plasma was nearly one-thousandth of that of an
ozonizer using the integrated number density of ozone
measured by UV absorption spectroscopy and the
contribution of UV radiation toward inactivation was not
dominant for P. digitatum inactivation by NEAPP.
Moreover, it is considered that the energy of charged
species is very low and densities are negligibly smaller
than those of neutral radicals in the remote plasma region.
Some paper reports that reactive oxygen species (ROS)
such as O or OH radical due to oxidation-decomposition
of cell membrane is important factor for the inactivation.[3, 4]
Fluorescence microscopy is powerful tool to investigate
the function of the cell and monitor the state of the cell.
On the plasma inactivation, in order to distinguish
between living and dead cell, CTG(cell tracker green),
PI(propidium iodide)， and dual staining (CTG + PI)
were used.[5,6] CTG is absorbed by all cells, but only
living cells transform it into fluorescent species. This
probe is used to verify cell viability: in living cells the
whole cytoplasm displays green fluorescence. PI penetrates only necrotic cells (with damaged membranes) and
bind to the DNA and RNA. It has not been directly
observed the destruction of the cell membrane by reactive species such as O or OH radicals.
In this study, we observed the spores of Penicillium
digitatum by using fluorescent microscopy in order to
investigate the effect of ROS. The spore inactivated by
the NEAPP was compared with that by the ultraviolet
sterilization lamp.
2
Experimental
Fluorescence image was observed by using a inverted
microscope (IX 70 by Olympus) with charge-coupled
device camera (DP 72 by Olympus). Cell tracker orange
stains living cells and the life time of the dye is longer
than 24 hours. The structure of cell tracker orange is
shown in Figure 1. When cell tracker orange stains living
cells, we can observe fluorescence of whole cell. When
the filtered light at the wavelength of 541 nm from the
mercury lamp irradiates, the cell tracker orange produces
fluorescence at 565 nm in the cells. Moreover, the diI
(1,1'-Dioctadecyl-3,3,Y,3'-tetramethylindocarboe-yanine
perchlorate) which is carbocyanine was used as a vital
fluorescence membrane dye.[8] The structure of diI and
phospholipid are shown in Figure 2 and Figure 3, respec-
tively. Octadecyl group of diI bind acyl group in the
phospholipid of the cell membrane, so that the membrane
is stained. The excitation wavelength of diI is 549 nm
and fluorescent wavelength is 565 nm.
A schematic diagram of non-equilibrium atmospheric
pressure plasma for the inactivation is shown in Figure 4.
In this system, an alternative voltage (AC) of 6 kV was
applied to two electrodes, and Ar gas of 3 slm was
flowed through the gap between the electrodes. The
distance between plasma and sample and exposure time
were 25 mm and 5 min, respectively. The power of
ultraviolet sterilization lamp, distance between plasma
and sample, and exposure time were 2.1 mWsec/cm2, 50
mm, and 5 min, respectively. All conditions are sufficient
to kill the spores of P. digitatum. The samples were
stained by cell tracker orange before the plasma treatment, while the samples were stained by diI after the
treatments of plasma and UV lamp.
Figure 1. structure of cell tracker orange.
CH3
3
CH3
CH3
CH3
CH
N+
CH
CH
N
CIO4-
CH3(CH2)17
(CH2)17CH3
Figure 2. structure of diI.
O
O
Results and discussion
Figure 5 fluorescence microscopic images of spores of
P. digitatum stained by cell tracker orange. Plasma
treatment time was changed. (1 : no treatment, 2 : 5 min,
3 : 10 min, 4 : 15 min.) Fungal spores of P. digitatum
were successful stained by cell tracker orange and whole
spore was stained by cell tracker orange. Fluorescence
area of spores became smaller with an increase in the
plasma treatment time. This result indicated that radical
produced from the plasma was induced to the spore and
react the spores.
Figure 6(a) shows fluorescence microscopic image of
the controlled Penicilium degitatum spore stained by diI.
It is confirmed that the membrane of Penicilium degitatum spore was successfully stained by diI. Fig. 6(b) and
Fig. 6(c) show the fluorescence microscopic images of
the Penicilium degitatum spore after the exposure by the
ultraviolet sterilization lamp and the plasma, respectively.
The organelle of some spores emits the fluorescent light
stained by diI in the case of the plasma treatment while
the fluorescent of the organelle by the UV lamp treatment
was not observed. For the living cell, the diI cannot be
penetrated to cell inside because the membrane has
selective permeability. On the other hand, the dose of the
UV light is sufficient to kill the spores under this experimental condition.
Table 1 shows the total number of the stained cell and
the number of the nucleus-stained cell. The stained rate
was calculated to be 44% for the plasma treatment while
the fluorescence from the nucleus was not observed on
the UV treatment. It is confirmed that the function of cell
membrane was inhabited by ROS produced from the
plasma. These results indicate that the membrane was
damaged by ROS and the diI penetrate into the organelle
through the damaged membrane.
Ar/O2 gas mixture ratio was changed on the experiment
using diI in order to investigate the effect of ROS. Figure
O
O
N+
P
O-
O
O
Figure 3. structure of phospholipid.
Power supply
Ar gas
Insulator
Distance L
Optical chopper
Deuterium
lamp
Quartz cell
Electrode
Plasma
Lens
Monochromator
Photomultiplier
Absorption path length
Sample
Figure 4 schematic diagram of non-equilibrium atmospheric pressure plasma for the inactivation.
Kershaw, G. A. Hidalgo-Arroyo, C. W. Penn, X. T. Deng,
J. L. Walsh, and M. G. Kong, Appl. Phys. Lett., 90,
073902 (2007).
[5] X. Deng, J. Shi, and M. G. Kong, IEEE Transaction
on plasma science, 34, 1310 (2006).
[6] I.E.Kieft, J.L.V.Broers, V. Caubet-Hilloutou, D. W.
Slaaf, F.C.S.Ramaekers, and E. Stoffels, Bioelectromagnetics, 25, 362 (2004).
[7] N. Yosida, H. Kano, S. Den and M. Hori, Extended
Abstracts, The Japan Society of Applied Physics and
Related Societies, 52, 190 (2005) (In Japanese)
[8] M. G. Honig and Richard I. Hume, J. Cell Biology,
103, 171 (1986).
Figure 5 fluorescent image of spores of Penicillium
digitatum stained by cell tracker orange. 1 : no treatment,
2 : 5 min, 3 : 10 min, 4 : 15 min.
7 shows the stained rate as a function of oxygen mixture
rate. Stained rate increased with an increase in the
oxygen mixture rate. Stained rate at oxygen mixture rate
of 0 % was about 40 %. The ROS was produced at 0 %
because the experiment was performed in the open air.
From these results, the function of cell membrane was
changed by ROS.
Conclusion
We observed the spores of Penicillium digitatum by
using fluorescent microscopy using two-type dyes in
order to investigate the effect of ROS. The spore inactivated by the NEAPP was compared with that by the
ultraviolet sterilization lamp. The function of cell membrane was changed by ROS produced from the plasma.
This work was partly supported by the Knowledge
Cluster Initiative (Second Stage) - Tokai Region Nanotechnology Manufacturing Cluster – and a Grant-in-Aid
for Scientific Research on Innovative Areas "Frontier
science of interactions between plasmas and
nano-interfaces" (No. 21110006) from the Ministry of
Education, Culture, Sports, Science and Technology in
Japan.
(a) control
4
5
References
[1]. A. Fridman, Plasma Chemistry (Cambridge University Press, New York, 2008).
[2] S. Iseki, T. Ohta, A. Aomatsu, M. Ito, H. Kano, Y.
Higashijima, M. Hori, Appl. Phys. Lett., 96, 153704
(2010).
[3] N. Hayashi, W. Guan, S. Tsutsui, T Tomari and Y.
Hanada, Jpn. J. Appl. Phys., 45, 8358(2006).
[4] S. Perni, G. Shama, J. L. Hobman, P. A. Lund, C. J.
(b) ultraviolet lamp
(c) plasma
Figures 6 fluorescent image of spores of Penicillium
digitatum.
Table 1. numbers of stained cell.
Plasma
treatment
UV
treatment
Total number
of cells
75
40
Number of
nucleus-stained cells
33
0
stained rate (%)
44
0
Stained rate[%]
100
80
60
40
20
0
0
20
40
60
80
O2 mixture rate [%]
100
Figures 7 stained rate as a function of O2 mixture rate.