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The efficacy of plasma-treated water as a root canal irrigant
Kaname Yamamoto, DMD (1), Tomoko Ohshima, DMD, PhD (2,3), Katsuhisa Kitano,
PhD (2,3), Satoshi Ikawa, PhD (4), Hiromitsu Yamazaki, DMD, PhD (2), Nobuko Maeda,
DMD, PhD (2), and Noriyasu Hosoya DMD, PhD (1)(1) Department of Endodontology, (2) Department of Oral Microbiology, Tsurumi University School of
Dental Medicine, Yokohama, (3) Center for Atomic and Molecular Technologies, Graduate School ofEngineering, Osaka University, Osaka, and (4) Technology Research Institute of Osaka Prefecture,
Osaka, Japan
Purpose: In recent years, attention has been drawn to disinfection techniques by plasma irradiation on humanbodies, however, the efficacy is not sufficient in the root canal. Therefore, the authors generated plasma-treatedwater (PTW) to concentrate the active species, and investigated the efficacy of PTW as a root canal irrigant.
Materials and Methods: PTW was produced using plasma prepared from helium containing nitrogen. Threetested microorganisms, Enterococcus faecalis, Candida albicans, and Candida glabrata, were selected, and their
growth with PTW was evaluated to determine minimum inhibitory concentration (MIC). Infected root canalmodels were created using 76 extracted human anterior teeth whose root canals were prepared and inoculatedwith one of the bacterial/fungal suspensions and cultured. A bacterial/fungal test of the root canals was
performed following PTW treatment. The effect of PTW was evaluated in an in situ model of infected root canalwith E. faecalis of 12 bilateral mandibular first molars of six 4-week-old male Wistar rats.
Results: PTW had a sterilizing effect against all tested microorganisms significantly. The MIC of PTW was adilution ratio of 0.125 against E. faecalis and a dilution ratio of 0.25 against C. albicans and C. glabrata. No
growth of microorganisms were observed in any of PTW experimental groups, that means the significant
efficacy of PTW in all test-infected root canal models in vitro and in situ of the rat.
Conclusion: PTW showed a sterilizing effect against E. faecalis, C. albicans, and C. glabrata, even in root
canals without obvious effect on oral mucosal tissue.
(Asian Pac J Dent 2017; 17: 23-30.)
Key Words: Candida, Enterococcus faecalis, plasma disinfection, root canal irrigation

Introduction
The mechanical cleaning is essential for root canal treatment. Traditionally, sodium hypochlorite has often beenused for chemical cleaning [1,2]. However, while sodium hypochlorite has a strong sterilization action, it alsostrongly irritates tissue and is associated with risk of injury caused by extravasation of sodium hypochlorite to
the periapical tissue, leakage of sodium hypochlorite into the oral mucosa, and dispersion of sodium
hypochlorite to skin and clothes [3,4]. To perform effective and safe treatment, a new root canal irrigant that isboth less harmful to living organisms and has sufficient sterilizing activity needs to be developed.
In recent years, attention has been drawn to plasma sterilization techniques for human body. Because various
active oxygen and nitrogen species can be produced from plasma generated, various types of plasma sterilization
is being reported [5-7]. Atmospheric pressure plasma in a state of thermal equilibrium can reach a very hightemperature, but it can be transformed to a state of non-equilibrium by subjecting it to pulse discharge, which
generates low-temperature atmospheric pressure plasma (LTAPP) that can avoid a thermal load. This LTAPP iscapable of irradiating living organisms in addition to sterilizing medical equipment. It is therefore expected to be
applied to treatments such as the promotion of burn healing or hemostasis, in addition to skin disinfection [8].
When applying LTAPP to living organisms, the plasma also needs to be effective against bacteria present influids because of the large amount of target bacteria present in damp environments. However, direct plasma
irradiation of bacterial cells present in fluids is difficult, and the sterilizing effect is limited. Ikawa et al. [9]therefore developed a “reduced-pH method” where the sterilizing effect against bacteria in fluid is markedly
enhanced by keeping the pH level of the fluid itself below 4.8. Research into the application of this technique is23

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already progressing even in the field of dentistry [10]. Caries-infected dentin is reported to reduce cariogenic
bacteria in dentin to a level below the detection limit [11]. The sterilization mechanism of direct plasma
irradiation is thought to result from HOO· generated by acid dissociation equilibrium of O2−·; however, there isconcern about the effect on the living organism in order for the simultaneously irradiated OH·, 1O2, and O tobring about a nonspecific chemical reaction.
The authors are continuing to conduct various studies with the objective of clinically applying LTAPP in thefield of dentistry and have reported that it is possible to produce plasma-treated water (PTW) with bactericidal
activity through active species by performing plasma irradiation of distilled water (DW) [12]. The properties of
PTW are similar to those of direct plasma irradiation, and PTW has strong bactericidal activity when the“reduced-pH method” is applied. Meanwhile, the half-life of PTW activity is relatively short, and the impact onliving organisms is considered very low. The half-life depends on the temperature; the half-life is several
seconds at 37˚C, several minutes at room temperature, several hours at 0˚C. However, when PTW is frozen at−80˚C, it can be semimaintained permanently [12]. When PTW is produced, it can be prolonged by cooling it in
ice cold water, which also allows the key active species to be maintained. PTW therefore has both extremely
strong bactericidal activity and a very small impact on living organisms, which makes the application of PTW todental treatment promising.
The purpose of this study was to investigate the efficacy of PTW when used as a root canal irrigant in
extracted human teeth and molars of living rat.

Materials and Methods
Generation of PTW
PTW was generated in the same way as research by Tasaki et al [13]. Helium containing several percent nitrogen
was used as the plasma generating gas. DW was poured into a quartz discharge tube (length: 1,000 mm, innerdiameter: 8 mm) to which electrodes were attached, and the plasma generating gas was simultaneously
controlled by mass flow controllers (Model 8500, Kofloc, Kyoto, Japan). Using a high-voltage pulse (20 kV, 10
kHz, 5-ns pulse width) generated by power supply (High Voltage Pulse Generator FPG 20-10NKN5; FID GmbH,
Burbach, Germany), atmospheric pressure glow plasma was generated through dielectric barrier discharge toirradiate DW. To ensure continuous generation of PTW and as long an exposure time as possible, the discharge
tube was placed at a slight angle. The equipment was totally cooled with ice cold water in an effort to avoid
losing PTW activity within the processing time. The generated PTW was immediately cooled with liquid
nitrogen and stored at −80˚C. Upon initiation of the experiment, the cryopreserved PTW was thawed withkeeping almost 0˚C while being stirred, after which 200 mM citrate buffer (pH 3.5) was added to adjust the pH
before PTW could be used.
Tested microorganisms and culture
The tested microorganisms were Enterococcus faecalis (ATCC19433), a bacterium, and Candida albicans(ATCC18804) and Candida glabrata (ATCC90030), two fungi. Cryopreserved E. faecalis suspension (500 µL)
was poured into 3 mL of brain heart infusion broth (BHI; Becton, Dickinson and Company, Franklin Lakes, NJ,
USA) and precultured for 24 hours at 37˚C under aerobic conditions, after which the concentration was adjusted
to 107-108 CFU/mL. Cryopreserved C. albicans and C. glabrata suspensions were each transferred by one
inoculation loop to 10 mL of tryptic soy broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with24

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5% dextrose (TSBD) for 24 hours preculturing at 30˚C under aerobic conditions, after which the concentrations
were adjusted to 107-108 CFU/mL.
Measurement of minimum inhibitory concentration (MIC)
Tested microorganism suspensions were distributed in 10 µL portions to a 96-well microtiter plate, to which 10µL of 200 mM citrate buffer (pH 3.5) and 80 µL of PTW were added. The samples were then kept for one
minute at room temperature, after which 100 µL of BHI were injected into the E. faecalis samples, and 100 µL
of TSBD were injected into the C. albicans and C. glabrata samples. The E. faecalis samples and C. albicans
and C. glabrata samples were subsequently subjected to 48 hours of aerobic culture at 37˚C and 30˚C,
respectively. Evaluation was done by observation of the presence or absence of microorganism growth andculture solution turbidity under room light. The tested PTW was observed at five different concentrations in a
two-fold serial dilution series. In the control groups, 90 µL of DW were added to samples, and four samples eachwere measured under each condition.
Creation of infected root canal models in vitro
The Ethical Review Board of Tsurumi University School of Dental Medicine (No. 865) gave its approval for thisstudy to be conducted. The tested teeth were 76 safely extracted human anterior teeth that had been stored insaline. The crowns were resected at a position 10 mm from the root apex, and the root canals were preparedusing hand files (Zipperer, München, Germany) up to size #60. During preparation, the root canals were also
irrigated frequently with 3% sodium hypochlorite. The root canals were immersed in DW in a 1.5 mL
polypropylene microtube, and sterilized with an autoclave. The root canals were then dried with a sterile paper
point, and #60 paper point impregnated with 10 µL of each suspension was inserted into the root canals
randomly divided into three groups. The E. faecalis group (n = 10) underwent aerobic culture within microtubes
for 24 hours at 37˚C, while the C. albicans (n = 16) and C. glabrata (n = 26) groups underwent aerobic culture
within microtubes for 24 hours at 30˚C.
Observation of the efficacy of PTW in infected root canal models
The infected models were stored before the experiment at 37˚C, and the experiment was subsequently conductedat room temperature. The paper points used for bacterial/fungal seeding were removed from the root canalmodels, after which a syringe mounted with a 27G needle was used to irrigate the models with 1 mL of PTW towhich 100 µL of 200 mM citrate buffer (pH 3.5) had been added. The needle was inserted until it was near theroot apex, after which the model was carefully irrigated while extracting bubbles. The root canals were left filledwith PTW for one minute, and PTW was removed with a paper point. A bacterial/fungal test was then conductedby using liquid medium. In other words, the #60 paper point was inserted into the root canal and impregnatedwith 20 µL of DW and then injected into a liquid medium after one minute. The C. glabrata group of models (n= 10) underwent one minute of prewashing with 1 mL of 200 mM citrate buffer (pH 3.5) before irrigating with
PTW, in addition to the procedure used in the aforementioned experimental group (n = 16). The control groups
were formed of a group irrigated with DW (n = 4) and a group of root canal models irrigated with citrate buffer(n = 4). The models underwent 48 hours culturing under aerobic conditions, after which the culture solution
turbidity was observed under room light.
Observation of the efficacy of PTW in situ models
The Animal Experiment Board of Tsurumi University School of Dental Medicine (No. 28A032) gave its
approval for this study to be conducted. The tested teeth were 12 bilateral mandibular first molars of six

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4-week-old male Wistar rats. After sedating the rats using ether, a triple anesthesia mix of 1.0 mg/mL
medetomidine hydrochloride (Domitor; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), 5.0 mg/mL
midazolam (Dormicum; Astellas Pharma Inc., Tokyo, Japan), and 5.0 mg/mL butorphanol tartrate (Betorphal;
Meiji Seika Pharma, Co., Ltd., Tokyo, Japan) was administered intraperitoneally at 0.1 mL/10 g [14]. The rats’jaws were held open with a mouth-opening device, and the access cavity was expanded with a round bur (0.8mm in diameter), after which the root canal was prepared using hand files (Zipperer, München, Germany) up to
size #15. Then, each root canal was injected with 10 µL of E. faecalis suspension using a micropipette, and thissuspension was made to fill the entire root canal by moving the file in a vertical up-and-down motion.
In each rat, one side was randomly assigned to the experimental group, while the opposite side was assigned
to the control group. The root canal was then irrigated with 1 mL of PTW or DW. After drying with a paper
point, bacterial test was conducted within the root canal by a simple culture method. In addition, changes in oral
mucosal tissue with which PTW came into contact were observed using a magnifying glass.
Statistical analysis
The efficacy of PTW in an infected root canal model and in situ model was statistically investigated between
conditions using chi-squared tests (p < 0.05).

Results
Measurement of MIC
PTW had a sterilizing effect against all tested microorganisms (Table 1). The MIC of PTW was a dilution ratioof 0.125 against E. faecalis and a dilution ratio of 0.25 against C. albicans and C. glabrata. Bacterial/fungal
growth was seen in all control groups.
Table 1 Minimum inhibitory concentration (MIC)
Tested microorganisms
E. faecalis
C. albicans
C. glabrata

Dilution rate of PTW
0.125
0.25
0.25

The efficacy of PTW in infected root canal models
The results are presented in Table 2. No growth of residual microorganisms was seen in any of the infected root
canal models in the E. faecalis and C. albicans groups, even without prewashing. In the C. glabrata group,
growth of residual microorganisms was seen in two samples in which prewashing with citrate buffer was not
performed; however, no growth was seen in any of the groups in which prewashing was performed. Growth wasseen in all control groups. The results of the statistical analysis showed significant efficacy of PTW in all
test-infected root canal models.
Table 2 Success rate of sterilization in the models of infected root canal *(%)
Tested microorganisms
E. faecalis
C. albicans
C. glabratawithout pre-wash
Control (DW)PTW
0 (n = 4)
100 (n = 10)
0 (n = 4)
100 (n = 16)
0 (n = 4)
87.5 (n = 16)with pre-wash
Control (Citric acid buffer)
0 (n = 4)
0 (n = 4)
0 (n = 4)PTW
Not doneNot done
100 (n = 10)*The rate of root canals which did not detect the microorganisms

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The efficacy of PTW in situ models
No growth of residual microorganisms was seen in any of the experimental groups of rat molars (Table 3).
Meanwhile, bacterial/fungal growth was seen in all control groups. The results of this statistical analysis showedsignificant efficacy of root canal irrigation with PTW in the experimental groups compared with the control
groups. In addition, no changes were seen in oral mucosal tissue that came into contact with PTW that
overflowed after root canal irrigation.
Table 3 Success rate of sterilization in the models of Wistar rats *(%)
Tested microorganisms
E. faecalisControl (DW)

PTW
without pre-wash
100 (n = 6)

0 (n = 6)

with pre-wash
Not done*The rate of root canals which did not detect the microorganisms

Discussion
The application of LTAPP to living organisms for skin disinfection and other purposes is being studied due tothe hypoallergenic nature of its sterilizing effect [6,7]. The sterilizing effect on intraoral microorganisms andapplication to the root canal of LTAPP are also being reported in the field of dentistry [15-18]. However, theseapplied studies involve direct irradiation with LTAPP, which makes practical application of LTAPP difficult forreasons including a required long irradiation time and the accompanying generation of ultraviolet light, heat, andharmful radicals. Meanwhile, Usui et al. [11] report achieving a dramatic sterilizing effect against bacteria incarious dentin in a short period of time by using direct plasma irradiation and the “reduced-pH method,” thereby
demonstrating the potential of plasma for clinical application. Prior to the present study, we therefore evaluatedthe in vitro sterilizing effect of direct plasma irradiation in E. faecalis infected root canals for the purpose of
applying plasma to root canal disinfection. When we directly irradiated infected root canal models filled withcitrate buffer with plasma in the direction of the tooth axis, we did not see a sufficient sterilizing effect against E.
faecalis within the root canals. This is likely because convection of active species is unlikely to occur within root
canals with a complex microstructure. In the application of plasma sterilization to root canal disinfection, clinicalapplication time, reliable bactericidal activity, and access to microstructures are all necessary. To solve these
problems, in the present study, the efficacy of plasma in root canal disinfection was investigated by using PTW
generated through the application of LTAPP as a root canal irrigant.
Three tested microorganisms were chosen: E. faecalis, C. albicans, and C. glabrata. E. faecalis is a bacteriumwith low drug susceptibility among intraoral microorganisms. It is frequently detected in infected root canals
with refractory apical periodontitis and is reported to be involved in the acute transformation of apicalperiodontitis [19]. To date, E. faecalis has often been used in studies of the sterilizing effect of plasma irradiation[17,18]. C. albicans is frequently detected within the oral cavity and is resistant to various agents for root canaltherapy including calcium hydroxide [20]. It has also been detected in infected root canals with refractory apicalperiodontitis with persistent percussion pain. C. glabrata is the next most frequently detected fungus in the oralcavity, and both fungi are typical oral fungi.
MIC measurements were performed to confirm the bactericidal activity of PTW against the tested
microorganisms and if this bactericidal activity is concentration-dependent. The results showed that PTW had asterilizing effect against all tested microorganisms (Table 1), and that this effect occurred at different

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concentrations between E. faecalis and Candidas. The reason for this difference is likely the larger number of C.
albicans and C. glabrata cells than E. faecalis, which require a larger amount of active species to achievesterilization. Yamazaki et al. also reported seeing greater resistance of C. albicans than E. faecalis in sterilization
by direct plasma irradiation [10]. Furthermore, superoxide dismutase is a known factor behind thedecomposition of active oxygen, and its production volumes and types are greater in eukaryotes than inprokaryotes, which likely explains the higher MIC of C. albicans and C. glabrata as compared with E. faecalis.
In creating the infected root canal models, the root canals were prepared up to size #60 in order to form root
canals that were as uniform as possible. Irrigation with PTW alone had an effect below the detection limit in all
E. faecalis and C. albicans groups (Table 2). The detection limit was set as the minimum concentration at which
growth was detectable. Pre-measured detection limit concentration was 4.9 CFU/10 µL for E. faecalis, 0.2
CFU/10 µL for C. albicans, and 0.6 CFU/10 µL for C. glabrata. Fungal growth was seen in two samples in theC. glabrata group that were only irrigated with PTW. PTW shows strong bactericidal activity under reduced pH
conditions, but its sterilizing effect can diminish when the pH rises as a result of the decalcification action of
citrate buffer on root canal dentin. Thus, when irrigating with PTW was performed after one minute of
prewashing with citrate buffer, there was an effect below the detection limit in all samples, even in the C.
glabrata group. C. glabrata is reported to have a lower susceptibility to azole antifungals than C. albicans;however, the detailed mechanism remains unclear [21]. The difference in susceptibility to PTW may also be
attributable to a similar mechanism.
The purpose of the in situ study using rat molars was to confirm the effects of PTW on microstructures inexperimental systems closer to clinical reality. The tested microorganism used was E. faecalis, and sufficient
bacterial seeding was achieved by moving the root canal preparation instrument vertically up and down. An
effect of irrigation with PTW below the detection limit was seen in all root canals, and no effect was seen in theoral mucosal tissue that came into contact with PTW during the experiment. It suggests that the residual activity
of PTW disappeared immediately.
In current clinical practice, chemical root canal cleaning requires removal of objects including hard tissue
shards resulting from root canal preparation, smear layers, and residual pulp tissue. Sodium hypochlorite is
typically used to dissolve soft tissue, while ethylenediaminetetraacetic acid (EDTA) is typically used to dissolve
hard tissue. Citrate buffer is added to PTW to adjust the pH. Citric acid has a chelating action and mineralsolubility, leading to reports of studies in which citrate buffer is applied to root canals to remove smear layers[22]. Due to the potential additional effect of citrate buffer, irrigating with EDTA or similar substances was not
performed in preparation of infected root canal models or rat molar root canals in the present study whenobserving the efficacy of PTW as a root canal irrigant. Reduction in efficacy of PTW in the C. glabrata group(Table 2) was thought to be attributable to the rise in pH that occurs as a result of the decalcification action of
citric acid. Thus, to achieve a reliable effect, prewashing with citrate buffer or irrigating with a sufficient amountof PTW is needed.
Sufficient caution also needs to be paid to the effect of PTW on healthy dentin and periodontal tissue during
root canal irrigation. Sodium hypochlorite, which is frequently used in clinical practice, is also reported to havean effect on dentin and periodontal tissue, and reports of accidents resulting from extravasation of sodium
hypochlorite outside of the apical foramen are not uncommon [4,23-25]. On the other hand, PTW has a strong
bactericidal activity and a short half-life of the active species that it contains, which suggests that it is a very safe

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root canal irrigant with low residual activity. This property of PTW, wherein it detoxifies within a short periodof time, which has not been present in conventional disinfectants, gives PTW a strong advantage as adisinfectant that can be applied to living organisms.
PTW used in the present study was confirmed to both have a sterilizing effect on microorganisms in the root
canal after about one minute of action and the ability to reach root canal microstructures. PTW can also becryopreserved at −80°C, which was additionally confirmed to have no impact on its sterilizing effect during use
after thawing [12]. Studies and the scope of clinical application are likely to expand, because PTW combines
both long-term storability and convenience when moved. A future study is needed to investigate the specificeffects of PTW on hard and soft tissues and to confirm the biological safety of PTW for practical application.
In conclusion, PTW generated using LTAPP exhibited a sterilizing effect against E. faecalis, C. albicans, andC. glabrata, which are sometimes detected in apical periodontitis. PTW also showed a sterilizing effect inmolars of rat. Moreover, the lack of any observed effect on oral mucosal tissue suggested that PTW is aneffective root canal irrigant.
Acknowledgments
The authors declare that there is no conflict of interest regarding the publication of this manuscript. This study was supported in part by aGrant-in-Aid for Young Scientists (B), from the Ministry of Education, Culture, Sports and Technology, and Japan Society for the Promotionof Science (JSPS), KAKENHI (No. 16K20471). The authors acknowledge financial support from the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT) of Japan in the form of a Grant-in-Aid for Scientific Research, Research (B) no. 23340176 and 15H03583,
and a Grant-in-Aid for Scientific Research on Innovative Areas “Plasma Medical Innovation” (25108505).

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Correspondence to:Dr. Kaname Yamamoto
Department of Endodontology, Tsurumi University School of Dental Medicine
2-1-3, Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
Fax: +81-45-573-9599 E-mail: yamamoto-kaname@tsurumi-u.ac.jp
Copyright ©2017 by the Asian Pacific Journal of Dentistry.

30

Accepted January 20, 2017.
Online ISSN 2185-3487, Print ISSN 2185-3479