Basic Research—Technology
Antibacterial Efficacy of a New Sonic Irrigation
Device for Root Canal Disinfection
Klaus W. Neuhaus, DMD, MMA, MAS,* Melanie Liebi, DMD,* Simone Stauffacher, DMD,*
Sigrun Eick, DMD,† and Adrian Lussi, DMD*
Abstract
Introduction: Passive ultrasonic irrigation (PUI) is the
most widespread method used to activate irrigation so-
lutions. Concerns have been raised that PUI is less effec-
R oot canal infections
tive in curved root canals and is not passive at all. Our
aim was to compare a novel passive sonic irrigation
(PSI) device (6000 Hz) with PUI and manual irrigation
(MI) with respect to their efficiency in removing different
endodontic microorganisms from curved and straight
root canals. Methods: We performed 2 experiments
as follows. In a 3-day infection model, we included 8
groups of single or dual microbial species that were
rinsed with 0.9% sodium chloride using PSI, PUI, or
MI. Colony-forming units (CFUs) were counted after in-
cubation, and log10 transformations were performed for
statistical comparisons. In a 21-d infection model, we
tested the same irrigation protocols on 4 groups of mi-
croorganisms and used 1.5% sodium hypochlorite as
an irrigant. Infection control samples were taken at
day 0, 3, 5, and 7 after treatment and were subsequently
reincubated. Results: Using sodium chloride as an irri-
gant, the amount of reduction in CFUs compared with
the negative control was approximately 3 log10 units
for PSI at 6000 Hz, 2 log10 units for PUI, and 1 log10
unit for MI. PSI reduced the microorganism CFUs signif-
icantly better than PUI. Using sodium hypochlorite led to
a significant reduction in microorganism CFUs even with
MI. After 3 days, compared with MI, microorganism re-
growth significantly reduced after PSI and PUI treat-
ment, but in these groups, in at least half of the
samples, microorganisms were detectable after 7 days.
Conclusions: PSI at 6000 Hz might be at least equal
to PUI with respect to reduction of the microbial load
in curved and straight root canals. (J Endod 2016;-
:1–5)
Key Words
Disinfection, oral bacteria, root canal, sonic irrigation,
ultrasonic irrigation
From the *Department of Preventive, Restorative and Pediatric Dentistry and †Laboratory of Oral Microbiology, Department of Periodontology, School of Dental
Medicine, University of Bern, Bern, Switzerland.
Address requests for reprints to Dr Klaus W. Neuhaus, Department of Preventive, Restorative and Pediatric Dentistry, School of Dental Medicine, University of Bern,
Freiburgstrasse 8, CH 3010, Bern, Switzerland. E-mail address: klaus.neuhaus@zmk.unibe.ch
0099-2399/$ - see front matter
Copyright ª 2016 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2016.08.024
JOE — Volume -, Number -, - 2016
are caused by a variety
of mainly anaerobic gram-
Significance
Passive sonic irrigation at 6000 Hz seems to
perform equal to or better than passive ultrasonic
positive bacteria (1). A
irrigation.
persistent intraradicular
presence of bacteria after
chemomechanical treatment is considered to be a possible cause of endodontic failure
(2). Although facultative anaerobes such as Streptococcus gordonii, Fusobacterium
nucleatum, and Actinomyces oris have been isolated in primary endodontic infections
(3), the persistence of Enterococcus faecalis (4) or Candida albicans (5) has been
associated with persistent periapical lesions and the need for endodontic retreatment.
Sodium hypochlorite is considered a suitable disinfecting irrigation solution, but it has
been shown that ultrasonic activation of sodium hypochlorite (NaCl) enhances its effec-
tiveness (6). Nowadays, passive ultrasonic irrigation (PUI) seems to be the predomi-
nant activation method for endodontic irrigation solutions (7). The main reason for
the additional effectiveness via ultrasound has been shown to be caused by acoustic
streaming effects that increase wall shear stress and enhance rupturing of intraradicular
biofilm (8). However, several limitations have been identified that impose procedural
problems when using ultrasonic activation. Wall contact with the oscillating instrument
dampens the energy and constrains the file movement (9). Therefore, in curved root
canals, ultrasonic instruments are less likely to oscillate freely. It has been shown
that even in straight root canals an ultrasonic instrument comes into contact with the
wall during at least 20% of the working time (10). Furthermore, although ultrasonic
irrigation instruments usually possess a noncutting design, they are made of a metal
alloy that is harder than root dentin, and, therefore, their use risks changing root canal
morphology. Consequently, Boutsioukis et al (10) suggested that PUI be replaced by
ultrasonically activated irrigation.
In order to avoid the detrimental effects caused by ultrasonic activation, activating
the irrigation solution with sonically driven noncutting plastic tips was suggested.
Among the devices using this technique, the EndoActivator (Dentsply, Tulsa, OK) device
appears to be the best-documented system (11). The principle is to use a polyamide tip
to activate the solution and thus to prevent active cutting of the root canal walls or open-
ing of the apical constriction. Passive sonic activation at low frequency was shown to be
inferior to PUI with respect to bringing irrigation solution to the apex in variously
tapered and curved canals (12). Furthermore, using simulated lateral canal models,
no difference in cleaning efficacy was detected between PUI and passive sonic activation
at low frequency (13). The maximum frequency of the aforementioned sonic irrigation
system was measured to be 190 Hz (14).
A New Sonic Irrigation Device 1
Basic Research—Technology
A novel sonic activation device has been developed that can be
coupled to an air scaler that operates at 6000 Hz. The aim of the present
study was to measure the bacteria-reducing effect of sonic activation at
6000 Hz in straight and curved root canals compared with PUI and
manual irrigation.
Methods
We performed 2 experiments.
1. We tested the irrigation effect alone using 0.9% (w/v) sodium
chloride as an irrigant in a short-term infection model.
2. We tested the additional effect of activation using 1.5% (w/v) NaOCl
as an irrigant in a long-term infection model.
Experiment 1
Selection and Preparation of Teeth. For the experiment, we
chose roots from maxillary premolars, palatal roots from maxillary
molars, and maxillary front teeth. The teeth were obtained from a
pool of extracted teeth stored in 1% chloramine solution. Informed
consent was obtained from the donors. According to the local ethics
committee, an approval for the irreversibly anonymized use of these
bypass products is not needed.
The teeth were stored in 3% (w/v) NaCl in order to remove the
periodontal tissue. After separating the roots, the curvature was radio-
graphically measured in 2 planes according to the method of Schneider
(15). The roots were grouped into curvatures <15 and >25 for
further experiments. The working length was established using a size
10.02 file and was defined to be 0.5 mm shorter than the length after
first apical visibility of the instrument. The roots were then negotiated
with a reciprocating instrument size 25.08 (RECIPROC R25; VDW, Mu-
nich, Germany). The rinsing solutions were 3% NaCl and 17% EDTA in
alternating mode. After closing the apex with composite (Telio CS Inlay;
Ivoclar Vivadent AG, Liechtenstein), the roots were stored in deionized
water until further use.
Microbial Contamination. Microbes were precultured for
18 hours and then suspended.
Before the experiment, the roots were placed in water and auto-
claved (121 C, 20 minutes [Laboklav ECO; SHB Steriltechnik, Detzel
Schloss, Germany]). After sterilization, the roots were inoculated for
3 days at 37 C under the conditions described later with 8 different mi-
crobes or combinations of microbes:
1. S. gordonii American Type Culture Collection (ATCC) 10558
2. A. oris ATCC 43146
3. Fusobacterium nucleatum ATCC 25586
4. S. gordonii ATCC 10558 and A. oris ATCC 43146
5. S. gordonii ATCC 10558 and F. nucleatum ATCC 25586
6. E. faecalis ATCC 29212
7. Candida albicans ATCC 76615
8. Clinical isolates obtained from a recurrent endodontic infection (6
species)
Groups 2, 3, 4, 5, and 8 were cultured under anaerobic con-
ditions, group 1 with 5% CO2, and groups 6 and 7 under aerobic
conditions. The microorganisms were cultured for 18 hours on
tryptic soy agar (TSA) plates with 5% sheep blood. The suspensions
of S. gordonii ATCC 10558 were prepared in 0.9% sodium chloride
(McFarland 4) and afterward diluted 1:9 with brain-heart infusion
broth (Oxoid, Basingstoke, UK) supplemented with 5 mg/L b-nico-
tinamide adenine dinucleotide (Sigma-Aldrich, St Louis, MO). The
suspensions of F. nucleatum ATCC 25586 and A. viscosus ATCC
43146 were prepared directly in Wilkins-Chalgren broth (Oxoid)
2 Neuhaus et al.
with b-nicotinamide adenine dinucleotide. Anaerobic bacteria and
streptococci were mixed at a ratio of 9:1.
Sterility was monitored at the beginning of the experiment with mi-
crobial sampling and subsequent culturing on TSA under respective
conditions. The nutrient broth was renewed every day.
Irrigation Protocols. The irrigation solution contained 0.9% (w/
v) sodium chloride. For each bacterial group, 10 roots were used in
each of the 4 following irrigation protocols, which consisted of 5
straight roots and 5 curved roots:
1. Test group (n = 10): passive sonic activation (EDDY polyamide tip,
VDW), 6000 Hz, coupled to an air scaler (SonicFlex, intensity mode
III; KaVo, Biberach, Germany), 3  20 seconds
2. Positive control (n = 10): passive ultrasonic activation
(IrriSafe + VDW Ultra, VDW), 3  20 seconds, power set at 20%
according to the manufacturer’s instructions
3. Negative control 1 (n = 10): manual irrigation (30-G endo-
irrigation needle KerrHawe SA, Bioggio, Switzerland), 6 mL
4. Negative control 2 (n = 10): no treatment
In irrigation protocols 1 and 2, the roots were irrigated with 1 mL
NaCl solution between the activation periods, and 1 mL NaCl solution
was used as a final rinse. For irrigation protocols 1 and 3, the instru-
ment/irrigation needle was placed at the working length; for irrigation
protocol 2, the instrument was placed 1 mm short of the working length.
Counting of Microorganisms. Sterile paper points (R25, VDW)
were placed for 30 seconds in the roots. These paper points were then
transferred into sterile tubes filled with 1 mL NaCl solution. After inten-
sive vortexing for 20 seconds and exposing to ultrasonication, 0.1 mL of
the solution was dispersed onto an agar plate. After incubation at the
respective conditions for 2 days (aerobes) or 7 days (anaerobes),
colony-forming units (CFUs) were counted.
Experiment 2
In contrast to the setup described previously, we did not differen-
tiate between curved and straight root canals in the second experiment.
We tested groups 4 to 7 in a long-term infection model. The duration of
incubation was 21 days under the same conditions as described previ-
ously. The irrigation protocols were the same as those described earlier,
with n = 6 for each irrigation protocol. However, the irrigation solution
in this part of the experiment was 1.5% (w/v) NaOCl followed by 1 mL
NaCl solution.
In order to control sterility throughout the entire duration of the
experiment, microbial samples were collected twice a week, cultured
for 48 hours at 37 C on TSA, and checked for contamination.
Immediately after irrigation, a microbiological sample was taken
as described previously. For 7 days, the root canals were filled again
with nutrient broth, and they were incubated under the respective con-
ditions at 37 C. After 3, 5, and 7 days, microbiological samples were
taken from the root canals. We performed a qualitative dichotomic anal-
ysis confirming the presence or absence of bacteria after 0, 3, 5, and
7 days of reincubation.
Statistics. All statistical results were calculated using R 3.1.0
(R-project; University of Vienna, Vienna, Austria) or SPSS 23 (IBM,
Armonk, New York).
For the first experiment, normality was checked by computing QQ
plots as well as P values according to the Shapiro-Wilk test (P = .0004,
indicating non-normality). Therefore, nonparametric methods were
applied.
The goal was to compare the log10-scaled number of microorgan-
isms after different treatment procedures. Secondly, the influence of the
JOE — Volume -, Number -, - 2016

covariable ‘‘curvature’’ was also analyzed. These analyses were
performed with a nonparametric analysis of variance (16).
P values were corrected for multiple testing with the Holm method.
Variables with significant effects in the nonparametric analysis of vari-
ance were tested afterward with Wilcoxon rank sum tests. These post
hoc tests were not corrected for multiple testing and were used explor-
atively.
For experiment 2, a global analysis was performed with the
Kruskal-Wallis test, and at each time point pair-wise comparisons
were performed with the Wilcoxon-Mann-Whitney test. The level of sig-
nificance was set to 0.05.
Results
The roots were not contaminated, and only the inoculated micro-
organisms were detected on plates throughout the entire experiment.
Experiment 1
All irrigation methods significantly reduced the number of micro-
organisms compared with the control group with no treatment. The irri-
gation methods were significantly different from each other. The
amount of reduction compared with the negative control was approxi-
mately 3 log10 units for passive sonic irrigation (PSI) at 6000 Hz, 2
log10 for PUI, and 1 log10 unit for manual irrigation. The exact CFU
numbers and the respective P values for the pair-wise comparisons
are provided in Table 1.
The covariable ‘‘curvature’’ had a highly significant impact on the
counts of microorganisms. Nevertheless, the number of remaining mi-
croorganisms after irrigation was significantly more reduced by PSI
than PUI both in straight and curved root canals (Fig. 1A and B).
Table 2 shows the median log10 CFUs differentiated by group of micro-
bial species and curvature.
Experiment 2
With NaOCl 1.5% (w/v) as the irrigant in the long-term experi-
ment, neither S. gordonii/A. oris nor S. gordonii/F. nucleatum were
detectable after irrigation with PSI or PUI. Both PSI and PUI were
less effective against E. faecalis and C. albicans; immediately after treat-
ment, all samples were negative, but after 3 days at least 50% of the sam-
ples were positive. There were no differences between PSI and PUI at the
time points of 0, 3, 5, and 7 days (Table 3).
Discussion
In this in vitro study, it was shown that activated irrigation as
achieved by a novel sonic irrigation instrument is efficacious in
TABLE 1. Median Log10 Colony-forming Units (Interquartile Range) after Different Irrigation Protocols
Microorganisms PSI
S. gordonii 2.78*,† (0.99)
A. oris 4.08† (0.36)
F. nucleatum 2.38† (2.07)
S. gordonii/A. oris 4.23† (0.45)
S. gordonii/F. nucleatum 3.76† (0.51)
E. faecalis 2.97*,† (0.4)
C. albicans 1.74† (0.94)
Retreatment isolates 3.46† (0.27)
MI, manual irrigation; PSI, passive sonic irrigation at 6000 Hz; PUI, passive ultrasonic irrigation.
Superscript symbols indicate pair-wise comparisons to the boxes to the right in the same row.
*P < .05.
†
P < .01.
‡
Nonsignificant.
JOE — Volume -, Number -, - 2016
Basic Research—Technology
removing different bacteria from root canals. Passive sonic activation
at 6000 Hz, when used without disinfecting irrigants, was even more
efficacious than ultrasonic activation. This finding was confirmed for
all strains of microorganisms used in this study, and the efficacy was
greatest with respect to the removal of C. albicans. Both bacteria that
are associated with primary endodontic infections and microorganisms
(E. faecalis and C. albicans) that are associated with the need for end-
odontic retreatment were included. The reduction pattern was consis-
tent for short-term incubation. We also showed that NaOCl is effective
against a number of microorganisms, even when applied with manual
irrigation; both PSI and PUI were able to significantly enhance the anti-
microbial effect.
Roots were separated (decoronated) to have similar conditions in
the laboratory. This can be regarded as a technical limitation in this
study because this allows easier access to the root canals, and there
is no coronal depot of irrigation solution.
Passive sonic activation of irrigation needles has been investigated
in previous studies. Little or no additional effect was found in compar-
ison with manual irrigation. In a study using simulated plastic root ca-
nals, no difference could be found between sonic activation and
ultrasonic activation in reducing bacterial counts (17). In addition, Tar-
divo et al (18) found no difference in antimicrobial activity against E.
faecalis when comparing ultrasonic irrigation and sonic irrigation in
extracted roots using NaOCl as the irrigant. Similarly, no difference
could be detected between sonic activation and manual irrigation
with 0.5% sodium hypochlorite in a clinical study (19).
By contrast, we found the highest reduction of microorganisms us-
ing a novel sonic irrigation device. One reason could be that the novel
device operates on a much higher frequency of 6000 Hz compared with
the maximum 170 Hz in the EndoActivator system. As mentioned earlier,
cavitation might occur in systems with frequencies above 30,000 Hz.
However, the effect of cavitation is not only dependent on the frequency
of the instrument inside the root canal but also on the amplitude of the
swinging instrument and thus might occur at lower frequencies (20).
Measurements have shown an amplitude of 11 mm for an ultrasonic in-
strument and 346 mm  41 mm for the novel PSI device used in this
experiment (20). Moreover, it can be suggested that the hydrodynamic
effects are of a different nature. The movement of the tip causes oscil-
lation of the fluid movement inside the root canal. This oscillation leads
to shear stress at the root canal walls, which includes high velocities and
high energies. Whether or not these energies from oscillating liquid are
high enough to tear apart bacterial cell membranes remains to be stud-
ied. Furthermore, there is probably an overlapping directional flow
because the system is closed at the apex and open at the root canal
entrance.
PUI MI Control
2.93† (1.3) 4.23† (0.47) 5.36 (0.67)
4.58† (0.33) 5.29† (0.22) 6.39 (0.23)
4.07*,† (1.85) 4.93* (1.52) 5.88 (1.35)
4.58† (0.28) 5.42† (0.45) 6.55 (0.28)
4.96‡,† (0.51) 4.99† (0.33) 6.65 (0.19)
3.84† (0.75) 4.68* (0.24) 6.47 (2.1)
3.26† (0.58) 3.68† (0.14) 4.67 (0.2)
3.96† (0.45) 4.64† (0.27) 5.88 (0.23)
A New Sonic Irrigation Device 3
Basic Research—Technology
A Straight canals tip can be observed in the visible strong liquid activation. Although the
latter device produces a tiny amount of aerosol, PSI at 6000 Hz visibly
8 **
** strongly activates the liquid and creates a constant amount of exceeding
** water droplets that is greater than the aerosol produced with PUI. Thus,
**
6 ** with PSI in this study, it was necessary to replenish the root canal drop-
** wise in order to avoid drying by strong liquid activation or overflowing
log10 cfu

of the irrigation solution. It could be argued that the greater effect of PSI
4
compared with PUI was because a larger amount of irrigation solution
was used and is inherent in methods that use continuous flushing versus
2 intermittent flushing. However, in the group with manual irrigation,
even more rinsing solution was used than in the PSI and PUI groups.
0
Therefore, the effect is likely attributable to hydrodynamic effects.
Control PSI PUI MI In this study, both straight and curved root canals were tested. The
irrigation methods
main obstacle of the efficacy of ultrasonic devices appears to be the cur-
vature of the root canals. It has been shown that cavitation effects occur
mainly at a freely oscillating tip of an ultrasonic instrument ‘‘depending
B Curved canals on the wall contact’’ (24). Consequently, because in curved root canals
8 ** it is usually not possible to avoid wall contact, the cleaning effect of PUI
**
** through cavitation is restricted (10). This has been shown in studies
** examining debris removal using ultrasonic irrigation. A less pro-
6 **
** nounced effect was found beyond the root canal curvature, whereas
in straight portions of the root canals debris removal was better (25,
log10 cfu

4 26). Still, any activation is better than no activation for cleaning the
2
0 manual irrigation in the short-term infection experiment. This was
Control PSI PUI MI
irrigation methods
Figure 1. Log10 CFUs after different irrigation protocols in (A) straight canals
and (B) curved canals. **P < .01.
Ultrasonic activation is supposed to support the endodontic
irrigation protocol. There are several possible reasons for the greater
effectiveness of PUI compared with manual irrigation. First, the irriga-
tion solution becomes activated because of the implosion of microcavi-
tation bubbles and hydrodynamic effects. PUI has been shown to clean
root canal walls from the smear layer, debris, and bacteria better than
manual irrigation (21). A second effect that has been described derives
from a heating effect in the root canal. Part of the energy is transformed
to heat energy, which leads to an intracanal temperature of 45 C (22).
The thermal effect might be advantageous when used in combination
with NaOCl (23). Whether or not PSI at 6000 Hz is able to heat the irri-
gation solution is the object of ongoing research.
Clinically, a difference between the novel sonic devices operating
at 6000 Hz compared with PUI with the IrriS (VDW, Munich, Germany)
apical portion of curved root canals (26). In our study, as expected,
‘‘curvature’’ was a significant variable for treatment outcome (reduction
of bacterial counts). However, even in curved canals, sonic activation at
6000 Hz performed significantly better than ultrasonic irrigation or
true for bacteria that are associated with primary endodontic infections
(groups 1–5) as well as for microorganisms associated with endodon-
tic retreatment.
This study also included a long-term experiment with 21 days of
root canal infection. The rationale was that after 3 days of incubation,
a young biofilm and planktonic microorganisms would be expected,
but after 21 days of incubation, many of the microorganisms might
be located within dentinal tubules. Thus, we wanted to measure the
in-depth cleaning effect by incubating the roots after the irrigation pro-
cedure for another 7 days in order to allow regrowth of microorganisms
that survived the cleaning procedure, presumably in dentinal tubules.
We wanted to follow a clinical protocol as closely as possible, which
is why only a final rinsing with 1 mL NaCl solution but no inactivation
of NaOCl with sodium thiosulfate was performed. Quantitatively, we
did not detect a statistical difference between PSI and PUI after irrigation
with NaOCl, but both methods performed significantly better than
manual irrigation in a long-term infection model.
The novel PSI device at 6000 Hz seemed to reduce bacteria at least
as well as PUI and thus might represent an interesting option for inclu-
sion into endodontic irrigation protocols. In a survey among American

TABLE 2. Median Log10 Colony-forming Units after Different Irrigation Protocols, Differentiated by Group of Microbial Species and Root Curvature
PSI PUI MI Control
Microorganisms Straight Curved Straight Curved Straight Curved Straight Curved
S. gordonii 2.78 2.96 2.92 3.72 4.25 4.03 5.04 5.24
A. oris 4.02 4.39 4.56 4.71 5.28 5 6.42 6.26
F. nucleatum 1.60 2.39 2.9 4.31 4.62 4.93 5.28 6.3
S. gordonii/A. oris 4.03 4.32 4.46 4.7 5.4 5.2 6.46 6.69
S. gordonii/F. nucleatum 3.65 3.8 5.08 4.84 5 4.98 6.58 6.7
E. faecalis 2.98 3.42 3.85 3.86 4.66 4.82 5.54 7.57
C. albicans 1.7 1.78 3 3.54 3.61 3.68 4.62 4.7
Retreatment isolates 3.45 3.51 3.85 4.26 4.53 4.66 5.89 5.78
Curved, curvature >25; MI, manual irrigation; PSI, passive sonic irrigation at 6000 Hz; PUI, passive ultrasonic irrigation.

4 Neuhaus et al. JOE — Volume -, Number -, - 2016

 Basic Research—Technology
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