Sensors and Materials, Vol. 24, No. 6 (2012) 311–322
MYU Tokyo
S & M 0887

Rapid Detection of Flavobacterium psychrophilum
Using Fluorescent Magnetic Beads
and Flow Cytometry
Kyoko Hibi, Yasutoshi Yoshiura1, Hideki Ushio,
Huifeng Ren and Hideaki Endo*
Faculty of Marine Science, Tokyo University of Marine Science & Technology,
4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan
1Aquatic Animal Health Division, National Research Institute of Aquaculture,
Fisheries Research Agency, 224-1 Hiruta, Tamaki, Mie 519-0423, Japan(Received July 25, 2011; accepted February 9, 2012)
Key words: fluorescent magnetic beads, immunomagnetic separation, fish disease,  Flavobacterium
psychrophilum, harmful bacteria, detection

Flavobacterium psychrophilum has emerged as one of the most significant bacterial
pathogens in salmonid aquaculture worldwide. We have been studying the detectionof harmful bacteria using immunomagnetic separation and flow cytometry (FCM).   Inthis study, we used fluorescent magnetic beads and 5-cyano-2,3-ditolyl tetrazolium
chloride (CTC).   Bacteria were specifically collected using fluorescent magnetic beads
with only one antigen-antibody reaction.  CTC turns into a red fluorescent formazan thatis detectable by FCM. F. psychrophilum cells were stained with CTC and labeled withfluorescent magnetic beads.  Double-stained bacteria (red fluorescence by CTC and green
fluorescence from fluorescent magnetic beads) were detected by FCM.   Bacterial cellnumbers were determined by FCM and compared with those measured by a traditional
colony counting method in the range of 102–108 cells/ml. The FCM assay could provide
a bacterial cell count within 1 min and the total assay time, including sample preparation,
was less than 3 h.

1.

Introduction

The ability to determine the number of viable bacterial cells in water samples is very
important in fishery industries, such as aquaculture, fish markets, and seafood products.  
Corresponding author: e-mail: endo@kaiyodai.ac.jp

*

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Measurements are made to determine total cell counts, total culturable cell counts, andthe presence of specific viable bacterial cells, such as pathogens.   After 20 years ofintensive research, the most effective method of determining whether bacterial cells arealive or dead remains controversial.(1) During the last two decades, various alternatives
have been pursued, such as real-time monitoring methods for assessing viability(2) andmethods to determine the existence of injured and ‘viable, but not culturable’ cells
exposed to stressors.(3,4)
Cell-specific assays to detect the respiratory activity of bacteria with the use ofdifferent tetrazolium salts have been developed.  Tetrazolium dyes are reduced from acolorless complex to a brightly colored, intracellular formazan precipitate by componentsof the electron transport system and/or a variety of dehydrogenase enzymes present in
active bacterial cells. Because electron transport is directly related to cellular energy
metabolism in respiring cells, the ability of cells to reduce tetrazolium compounds is
considered to be an indicator of bacterial activity. An alternative approach is the useof the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC).   CTC is reducedby bacteria to a water-insoluble, red fluorescent formazan product, which allows forthe quantification of the metabolic activity of bacteria under both aerobic and various
anaerobic conditions.(5,6) Several factors, however, can affect CTC reduction during
incubation.(7–9)   Although CTC counts are commonly determined by flow cytometry(FCM),(10–12) epifluorescence microscopy can also be used to overcome the rapid fadingof the fluorescence signal,(13) yielding equal or higher counts.(14)
FCM has become more popular and offers the ability to analyze individual cells
at numbers that are more representative of nature.(15) FCM allows for the rapid andsimultaneous measurement of various cell parameters with a precision of a few percent.
FCM is not adapted to detect event rates, however, because it is difficult to detect
fewer than 100 cells/ml, which is still 10- to 100-fold better than current microscopy
procedures. Thus, concentration technology is needed to increase the density of the cells.
One alternative method for rapidly concentrating samples uses antibody-coatedmagnetic beads and immunomagnetic separation (IMS) to concentrate target bacteria thatare then measured using FCM or another device.(16–18)  IMS with a specific antibody is a
powerful method of detecting target microbes from heterogeneous microbial suspensions.
IMS depends on the interaction between antibodies bound to cell surface antigens andparamagnetic beads. Samples drawn and collected by IMS are concentrated. Whenusing IMS, the resulting sample concentration can be tuned. An advantage of IMS is tobe able to wash bacteria collected once. It is because impurities are removed by washing
the sample.   We have quantified harmful bacteria using immunomagnetic beads andfluorescent dye-labeled antibody.(19,20)   The antibody conjugated to the fluorescent dye
is used to specifically label the target bacteria.   The magnetic bead, which is attachedto the antibody, is then used to isolate the target bacteria from the rest of the sample. A
concentrated sample of the target bacteria is thus produced.
Flavobacterium psychrophilum causes a severe disease in fish, particularly farmed
rainbow trout (Oncorhynchus mykiss), throughout the world. It can be transmitted
horizontally indirectly through the water and directly by fish-to-fish contact.   Over the
past 10 years in many parts of Europe, rainbow trout fry syndrome and bacterial cold

Sensors and Materials, Vol. 24, No. 6 (2012)313

water disease caused by F. psychrophilum have become the most serious bacterial fish
diseases. Current treatments of this problem include raising the water temperature andadministering medicine. The disease should be treated as early as possible to prevent ordelay the spread.
The purpose of the present study was to develop a method of rapidly detecting
viable bacteria using a combination of CTC, fluorescent magnetic beads, and FCM.  Thebacterial viability can be determined using CTC and the magnetic beads to be isolated.
Then the measurement sensitivity is improved by collecting bacteria of the target and the
FCM allows us to determine how much F. psychrophilum is in the sample.

2.

Materials and Methods

2.1

Materials

2.2

Microorganisms and culture

2.3

CTC staining conditions

The polymer sample used in this study was commercial water-soluble carbodiimide(WSC) and CTC obtained from Dojindo (Kumamoto, Japan). Tryptone and yeast
extract were obtained from Difco Laboratories (Detroit, MI). Sodium acetate andcalcium chloride were obtained commercially and were of analytical reagent grade.
Bonito extract was obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan).
Fluorescent magnetic beads were obtained from Chemicell (Berlin, Germany). Microcon
was obtained from Millipore (Billerica, MA). Rabbit anti-F. psychrophilum serum was
manufactured by Kitayama Labs Co. Ltd (Nagano, Japan).
F. psychrophilum FPC 840, Escherichia coli IM 12119, Pseudomonas fluorescens
IAM12022, Lactococcus garvieae H 9507, Staphylococcus aureus, and Vibrio sp. wereused as model microorganisms. F. psychrophilum FPC 840 was cultured in a cytophaga
broth containing 0.5 g/l tryptone, 0.5 g/l yeast extract, 0.2 g/l beef extract, and 0.2 g/l
sodium acetate trihydrate, at pH 7.2 at approximately 15°C for 3 d. E. coli, P. fluorescens, S.
aureus, and L. garvieae were cultured overnight in Luria Bertani medium consisting of
10 g/l tryptone, 5 g/l yeast extract, and 5 g/l NaCl using a shaking flask at 25°C for 20 h.
These studies were performed with cultures of the above-mentioned strains grown incytophaga broth (15°C, 48 h) and washed in phosphate buffer (PB; 10 mM Na2HPO4, 10
mM NaH2PO4, pH 7.2). A 20 mM CTC stock solution in distilled water was preparedand stored at 4°C until use. F. psychrophilum was washed in PB, and after discarding
the supernatant solution, equal amounts of PB and medium were added to bring theconcentration of CTC to 0.2 mM.   The reduction of CTC to the fluorescent formazan
was monitored by FCM after incubating the bacteria at 15°C and agitating with either arotating cultivator (Rotate RT-50, Koshigaya, Japan) or a micromixer (Micromixer E-36,
Koshigaya, Japan). Samples of the culture were obtained every 30 min for 3 h. The
percentage of cells with reduced CTC was also determined for cultures containing no
medium (starved cultures).

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2.4

Preparation of fluorescent magnetic bead-antibody conjugates

2.5

Flow cytometry analysis

2.6

CTC-fluorescence double staining of magnetic beads

The antibodies were ultrafiltered to facilitate an efficient reaction between the
antibodies and magnetic beads. A sample solution containing anti-F. psychrophilum
rabbit IgG antibody (200 µl) and WSC buffer (100 µl) were added to a filtration
tube. WSC was used as a cross-linking agent. This mixture was mixed thoroughly by
pipetting and centrifuged at 8,000 g for 10 min. More WSC buffer (100 µl) was added
to the filtration tube and the mixture was centrifuged again at 8,000 g for 10 min.  Thefluorescent magnetic bead (10, 50, 100, 150, and 200 ml) solution was added to the
IgG antibody concentrated in the filtration tube.   The concentration of the fluorescentmagnetic beads was 2.25 g/cm3. The magnetic beads are coated in such a way that the
carboxyl group is free to react with the amino group of the antibody and the WSC buffer.
The carbodiimides react with the terminal carboxylate-groups of the magnetic beads
to form highly reactive O-acylisourea derivatives, which then readily react with theamino groups of the ligands. This binary covalent binding system thus guarantees good
reproducibility of the immobilization.  This sample was incubated at 37°C for 2 h after
mixing thoroughly by pipetting. WSC buffer (190 µl) was added to the sample and thesample was stored at 0 to 5°C.
A Beckman Coulter Epics XL flow cytometer equipped with an air-cooled 488 nm
argon-ion laser at 15 mV was set up with the standard configuration (Beckman Coulter,
Fullerton, CA). Two fluorescence intensities (FL) were measured simultaneously.
Fluorescence emission was measured in four channels: FL1, wavelength 490–550 nm;
FL2, 570–580 nm; FL3, 600–620 nm; and FL4, 665–685 nm. The green emission (FL1)from the fluorescent magnetic beads was collected with a 525 nm band-pass filter.  Thered emission (FL4) from CTC was collected with a 675 nm band-pass filter.  The stoptime for the passage of the sample was set at 60 s, and the event number (number ofparticles passing through the laser beam) was measured as the number of bacteria.
The function of “Compensation” is provided in FCM. In the FCM determination, theemission spectra from the two fluorescent probes overlap, thus requiring compensation.   
Spectral overlap between green fluorescence and red fluorescence produces light that is
detected by both FL1 and FL4 detectors.  The amount of green fluorescence detected by
the FL4 detector (i.e., the red fluorescence detector) was regarded as excess fluorescenceand was subtracted from the signal. This amount of spectral overlap was correctedby subtracting the percentage from the total green fluorescence pulse generated by theFL1 detector.  Therefore, the green fluorescence produced by the fluorescent magnetic
beads detected by the FL4 detector was considered to be interference. By subtracting
a percentage of the total green fluorescence pulse from the total pulse generated by theFL4 detector (CTC plus fluorescent magnetic bead fluorescence), we obtained a redfluorescence-only pulse.  Thus, a double measurement of the particle with both red andgreen fluorescence was prevented.
Cells with red fluorescence from CTC were determined by FCM to confirm that thecells were stained. Magnetic beads prepared beforehand (5, 10, 30, 50, 70, and 100 µl)

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Sensors and Materials, Vol. 24, No. 6 (2012)were added to the stained cells. The cells were mixed well and incubated at room
temperature for 10, 20, 40, 60, or 80 min.   The antibody-coated fluorescent magnetic
beads and F. psychrophilum cells were antigen-antibody reacted. After washing twice
to avoid nonspecific binding of cells, the magnetically coated cells were immobilizedwith the magnet and resuspended in PB. A total of 1 ml of F. psychrophilum suspension
containing 104 to 108 cells/ml was isolated and then added to 1 ml of PB, and 20 ml of
101 to 105 cells/ml F. psychrophilum suspension was isolated and then added to 200 µl of
PB. High concentrations (104 to 108 cells/ml) of F. psychrophilum were not concentrated,
and low concentrations (101 to 105 cells/ml) of F. psychrophilum were concentrated
100-fold. These cell suspensions were used for the FCM analyses.

3.

Results and Discussion

3.1

Relationship between CTC staining condition and time

The frequency of cells containing reduced CTC was dependent on the probe
concentration and nutrient addition.  The CTC concentration was adjusted to 2 mM (final
concentration), on the basis of the report of Yamaguchi et al.(12) For E. coli cultures,
tryptone soya broth was used for CTC assays,(13) but we chose cytophaga broth, whichis the medium used for F. psychrophilum. The relationship between CTC staining
condition and time is shown in Fig. 1. The staining rate increased with an increase intime under all three conditions. When the micromixer was used to agitate the culture,
the staining rate reached 94.9% at 120 min and 96.8% at 180 min. The staining rate
reached a plateau by 120 min (Fig. 1). The best CTC staining condition was considered

CTC staining cells (%)

100
80
60

40
20
0
0

30

60

90

120

150

180

Time (min)Fig. 1. CTC staining conditions and time. Circles are for CTC staining ratio for bacteria
incubated with 600 rpm agitation by a micromixer every 30 min for 3 h. Triangles are for CTC
staining ratio for bacteria incubated with 10 rpm agitation by a rotator every 30 min for 3 h.
Crosses are for CTC staining ratio for bacteria incubated with standing culture every 30 min for 3 h.

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to be agitation with the micromixer for 120 min. The F. psychrophilum concentrationof the sample used for this experiment was 106 CFU/ml (CFU: colony forming units).
When a lower concentration of bacteria was stained with CTC, the ratio of stained cells
decreased somewhat (data not shown). CTC is reported to have toxic effects on bacterial
metabolism.(21) Although sodium chloride has been used in CTC assays of Lactobacillus
sakeii and Listeria monocytogenes cultures,(22,23) F. psychrophilum does not grow in
medium containing 2% salt.(24) Therefore, we did not add NaCl to the medium for the
CTC assay of F. psychrophilum. When the bacteria were killed using formalin, CTC
did not induce a red fluorescence.  Therefore, the CTC assay allowed us to distinguishbetween viable and dead F. psychrophilum.

3.2

Fluorescence scattergrams for F. psychrophilum

Green fluorescenceby fluorescent magnetic beads

F. psychrophilum cells suspended in PB were analyzed by FCM after staining with
CTC [Figs. 2(b) and 2(d); red fluorescence] and/or fluorescent magnetic beads [Figs. 2(c)and 2(d); green fluorescence].  Results for unstained F. psychrophilum measured by FCM
are shown in Fig. 2(a).  Cells stained with only CTC exhibit only red fluorescence.  In
the FCM determination, the emission spectra from the two fluorescent probes overlap,
thus requiring compensation.   Spectral overlap between green fluorescence and red

a

b

c

d

Red fluorescence by CTC
Fig. 2. Flow cytometric analysis of fluorescence staining cells and fluorescent magnetic beads.  
(a) F. psychrophilum cells suspension, (b) CTC stained cells, (c) fluorescent magnetic beads, (d)fluorescent magnetic beads and CTC double-stained cells.

Sensors and Materials, Vol. 24, No. 6 (2012)317

fluorescence produces light that is detected by both FL1 and FL4 detectors.  The amountof green fluorescence detected by the FL4 detector (i.e., the red fluorescence detector)
was regarded as excess fluorescence and was subtracted from the signal.  This amountof spectral overlap was corrected by subtracting the percentage from the total green
fluorescence pulse generated by the FL1 detector.   Therefore, the green fluorescenceproduced by the fluorescent magnetic beads detected by the FL4 detector was considered
to be interference.   By subtracting the percentage of the total green fluorescence pulse
from the total pulse generated by the FL4 detector (CTC plus fluorescent magnetic beads
fluorescence), we obtained a red-fluorescence-only pulse.  After measuring CTC-stained
bacteria and fluorescent magnetic beads alone, a “compensation system” for the FCM
was applied, and the signals shown in Fig. 2(d) were calculated. CTC-stained bacteria
were added to the fluorescent magnetic beads.   Fluorescent magnetic beads with theantigen-antibody were reacted with F. psychrophilum because the magnetic beads wereconjugated with anti-F. psychrophilum antibody. After the reaction, a neodymium magnet
was placed near the sample. The sample solution was reddish brown owing to the
mixture of CTC-stained bacteria and fluorescent magnetic beads.  When the neodymium
magnet came close to the sample, the reddish-brown particles were drawn to the magnet,
and the solution became transparent. The supernatant of this sample was discarded, andafter washing twice with PB, PB was added to the sample, and the sample was measured
by FCM [Fig. 2(d)].  There were cells with both green and red fluorescence.  There wereparticles in the area with only green fluorescence, which represent fluorescent magnetic
beads that did not react with the cells. These results indicated that F. psychrophilum
could be measured using fluorescent magnetic beads.  

3.3

Antibody specificity

3.4

Determination of optimal amount of antibody

The specificity of this method using CTC staining and fluorescent magnetic beads
was also examined for E. coli, P. fluorescens, L. garvieae, S. aureus, and Vibrio sp. (Fig. 3).
The concentration of each species was 106 CFU/ml. These bacteria were stained with
CTC in accordance with previous reports.(6)  After confirming that the cells were stainedusing FCM, the fluorescent magnetic beads were added.   The cells were magneticallyseparated with the neodymium magnet, and CTC-stained bacteria that had not boundto the magnetic beads were washed out. The antibody reacted with F. psychrophilum,with 90% of the cells being detected, but it did not react with E. coli, P. fluorescens, L.
garvieae, or S. aureus; less than 8% of these cells were detected.
CTC-stained bacteria (F. psychrophilum) were added to fluorescent magnetic beads
that were not conjugated with antibodies and then incubated for 1 h. Most of the CTCstained bacteria were washed out of the sample because there was no antibody. A small
number of bacteria were still detected, however, owing to the nonspecific adsorption of
the bacteria to the fluorescent magnetic beads (data not shown).   Washing the sample
twice was critical to prevent the nonspecific adsorption of the bacteria.  
The amount of antibody added to the magnetic beads was examined (Fig. 4). The
antibody was conjugated with fluorescent magnetic beads to determine the amount of318

Sensors and Materials, Vol. 24, No. 6 (2012)

Dual staining cells (%)

100
80
60

40
20
0

FCM event number (cells/min)Fig. 3. Antibody specificity to F. psychrophilum and other bacterial species. Each cell concentration
was prepared to 1 × 106 – 6 × 106 cells/ml. The FCM assay conditons were the same as in the caseof Fig. 1.

105

104

103

102

0

50

100

150

200

250

Amount of fluorescent magnetic beads
with antibody (µl)

Fig. 4. Effect of amount of antibody on bacterial detection (n = 3). The FCM assay conditions
were the same as in the case of Fig. 1.

bacteria. Different amounts of antibody (10, 50, 100, 150, and 200 µl) were added to
the fluorescent magnetic beads.   The concentration of the antibody solution was 0.5
mg/ml. Immunomagnetic beads conjugated with antibody were reacted with the F.
psychrophilum suspension. When the amount of antibody was 10 or 50 µl, the results of
the FCM measurements were low.  We assumed that not all fluorescent magnetic beads
were conjugated with the antibody because the amount of antibody was smaller than that

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Sensors and Materials, Vol. 24, No. 6 (2012)of the magnetic beads. When the amount of antibody exceeded 100 µl, the results of the
FCM measurements reached a plateau, indicating that the maximum number of beads
had conjugated with the antibody. The optimal amount of antibody was determined to be
100 µl.

3.5

Determination of optimal amount of antibody-conjugated magnetic beads

FCM event number (cells/min)

The effect of various amounts of fluorescent magnetic beads conjugated with theantibody on the FCM detection of bacteria was examined (Fig. 5). Different amountsof immunomagnetic beads conjugated with the antibody (5, 10, 30, 50, 70, and 100 µl)
were added to the F. psychrophilum suspension. The concentration of bacteria used was
106 CFU/ml. When 5 µl of immunomagnetic beads was added, the results of the FCM
measurements were low. We assumed that not all of the bacteria had reacted with themagnetic beads because the amount of immunomagnetic beads added was less than thatof bacteria. When we added 10 µl of the immunomagnetic beads, the results of the FCM
measurements increased. The response reached a plateau at 10 µl. The optimal amountof immunomagnetic beads used was 10 µl.
We added magnetic beads (10 ml) to bacteria of 106 CFU/ml to examine how
many bacteria react with one bead. The suspension of post-IMS magnetic beads andsuspension with papain added to the post-IMS magnetic beads were each transferred
onto plates to determine the number of CFUs by plate counting. Suspension with papain
buffer (concentration of 1.08 mg/ml) added to the post-IMS magnetic beads had twice
as many colonies than the suspension of only post-IMS magnetic beads. Papain buffer
helped the antigen detach from the antibody.(24) Therefore, about one or two bacteria arereactive to one magnetic bead.

105

104

103

102

0

20

40

60

80

100

120

Amount of fluorescent magnetic beads
conjugated to antibody (µl)

Fig. 5. Effect of fluorescent magnetic beads conjugated with antibody on bacterial detection (n = 3).
The FCM assay conditions were the same as in the case of Fig. 1.

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3.6

Determination of optimal incubation time

3.7

Correlation between FCM and colony-counting methods

The antigen-antibody reaction time of the immunomagnetic beads and bacteria was
examined (Fig. 6). Immunomagnetic beads (10 µl) were added to a 1 ml sample of F.
psychrophilum, and the mixture was incubated for 10, 20, 40, 60, or 80 min. Figure 6
shows that the FCM event number increased with an increase in time within the rangeof 0 to 40 min. When the incubation time exceeded 40 min, the FCM count reached aplateau and the counts at 60 and 80 min were almost the same. Therefore, the incubation
time was set at 60 min.

FCM event number (cells/min)Suspensions of F. psychrophilum cells at 102 to 108 CFU/ml were incubated with
CTC.  CTC-stained cells were not washed, and a 10 µl solution containing fluorescentbeads was added. After incubation for 60 min while standing at room temperature,
separated and washed cells were resuspended in PB. Only magnetic separation was used
for the high-concentration samples (105–108 CFU/ml; high concentration). After themagnetic separation, the supernatant was discarded while the bacteria were immobilized
by the magnet, and an amount of PB equal to that of the discarded solution was added.
Therefore, there was no change in the sample concentration. Magnetic separation andconcentration were applied to the low-concentration samples (102–105 CFU/ml; lowconcentration). After the magnetic separation, the supernatant was discarded while
the bacteria were immobilized with the magnet, and PB was added.   The amount of
PB added was 1/100 of the amount of the discarded sample. The concentration of thesample was therefore increased 100-fold. Figure 7 shows the measurement results ofthe prepared sample. In the high-concentration samples, magnetic separation alone was
useful for isolating the cells. When the cells were not concentrated, however, densitiesof less than 104 CFU/ml could not be measured. When the cells were concentrated,

105

104

103

102

0

20

40

60

80

100

Incubation time (min)Fig. 6. Effect of incubation time on bacterial detection (n = 3). The FCM assay conditions were
the same as in the case of Fig. 1.

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Cell number of bacteria (CFU/ml)
FCM method

Sensors and Materials, Vol. 24, No. 6 (2012)
107

104

a

b

103

106

102
105

10
y=407.0x0.327 R= 0.9923

104

103

104

105 106 107 108

Cell number of bacteria (CFU/ml)
Colony counting method1
109 10y=13.37x0.332 R= 0.957

102

103

104

105

106

Cell number of bacteria (CFU/ml)
Colony counting method

Fig. 7. Correlation between the proposed FCM method and colony counting method. (a)High concentration 104–108 CFU/ml. (b) Low concentration 102–105 CFU/ml. The FCM assay
conditions were the same as in the case of Fig. 1. CFU: colony forming units.

however, detection over a wide range of densities was possible. There was a strong
correlation between the values determined by the FCM method and by the colonycounting method for both the low and high concentrations (low concentration R = 0.9846;
high concentration R = 0.9923).
The combination of the CTC fluorescence probe with the fluorescent magnetic beads
allowed for the quantification of harmful viable bacteria.  It should also be possible to use
magnetic beads with different fluorescent properties conjugated to different antibodies
to measure two viable bacteria. This method can be used in environmental studies aswell as in various medical fields and the food industry.  For example, this method can beapplied to the problem of ballast water contamination, which is an international problemof marine pollution caused by ballast water and tank sediments.

4.

Conclusions

We determined the optimum conditions for CTC staining of F. psychrophilum todistinguish between viable and dead F. psychrophilum. Furthermore, we developed
a new assay method for the rapid determination of viable F. psychrophilum. The
calibration curve for F. psychrophilum was linear in the ranges of 105 to 109 and 102
to 105 CFU/ml.   In this assay method, a CTC-fluorescent probe and an IMS techniquewith fluorescent magnetic beads combined with FCM were used to detect viable F.
psychrophilum.

Acknowledgments
This research was supported in part by a Grant-in-Aid for Scientific Research (B)from The Ministry of Education, Culture, Sports, Science and Technology.

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