Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Efficacy of endodontic applications of ozone and low-

temperature atmospheric pressure plasma on root canals
infected with Enterococcus faecalis
€ reyen Kaya1, A.D. Kececi1, H.E. Gu
B. U € lda,s 1, E.S. C € ztu
, etin2 , T. O € ksuz 3 and F. Bozduman3
€ rk2, L. O
1 Department of Endodontics, Faculty of Dentistry, Su€leyman Demirel University, Isparta, Turkey
2 Department of Microbiology, Faculty of Medicine, Su€leyman Demirel University, Isparta, Turkey
3 Department of Physics, Faculty of Science & Arts, Su€leyman Demirel University, Isparta, Turkey

Significance and Impact of the Study: The present study handles different perspectives on chemome-
chanical preparation of root canals. Ozone and low-temperature atmospheric pressure plasma (LTAPP)
were investigated to determine whether they could be an alternative for NaOCl. Up to now, chemical
solutions (NaOCl, chlorhexidine digluconate, etc.…) have been used to disinfect the root canals. When
the reported effects of LTAPP on biological and chemical decontamination were taken into consider -
ation, a question rose whether it has antimicrobial efficacy in root canals infected with E. faecalis.
According to the findings of the present study, LTAPP may constitute a promising aid in endodontics in
disinfection of root canals.

Keywords
disinfection, Enterococcus faecalis, Low-
temperature atmospheric pressure plasma,
NaOCl, ozone.
Correspondence
Bulem U € reyen Kaya, Su €leyman Demirel U€ niv-
€
ersitesi, Di,s Hekimliği Fakultesi, Endodonti
AD, 32260 Kampu€s-Isparta, Turkey.
E-mail: bureyen@hotmail.com
2013/0822: received 26 April 2013, revised
16 August 2013 and accepted 17 August
2013
doi:10.1111/lam.12148
Abstract
This study aimed to compare the antimicrobial efficacy of low-temperature
atmospheric pressure plasma (LTAPP) design and gaseous ozone delivery system
with 2 5% NaOCl· on Enterococcus faecalis in root canal walls and dentine tubules.
The samples were divided into LTAPP (n = 12), ozone (n = 12), NaOCl
(positive control, n = 12) and saline (negative control, n = 6) groups. Microbial
samples were collected using paper points and dentin chips from root canals.
Antimicrobial efficacy was assessed by counting the colony- forming units of Ent.
faecalis before and after each irrigation protocol. Data were analysed using
Kruskal–Wallis, Wilcoxon signed-rank, Friedman and Bonferroni t (Dunn’s test)-
tests (P = 0·05). The microbial sampling with paper points showed antibacterial
efficacy of NaOCl, LTAPP, ozone and saline in descending order, respectively (P
< 0·05). The microbial sampling with dentin chips demonstrated a superior
efficacy of LTAPP compared with NaOCl in the middle third (P < 0·05), while
both had similar effects in coronal and apical thirds (P > 0·05). NaOCl and LTAPP
were better than ozone at the coronal and middle parts of the root canals (P <
0·05). These findings led us to suggest that LTAPP, which has no thermal and
chemical effects, may be of great aid in endodontic treatment.

canal infections in its planktonic or biofilm forms (George

Introduction
The complex morphology of root canal systems with variety
of dentin tubule morphologies, apical canal ramifi- cations,
isthmuses and irregularities, where bacteria may exist in the
form of biofilms, makes the elimination of microbiota
challenging (Nair 2006). Enterococcus faecalis is the
representative micro-organism of secondary root
et al. 2005), and studies related to the elimina- tion of root
canal infections have been designed by using Ent. faecalis
biofilm models (Spratt et al. 2001; Chavez De Paz 2012).
Although NaOCl, as endodontic irrigant, has a superior
antibacterial efficacy and is accepted as ‘gold standard’
(Garcia et al. 2010), its cytotoxic properties force the

8 Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology
 Efficacy of Ozone and LTAPP on Ent. faecalis
researchers to innovate the current irrigation regimen. Low-
temperature atmospheric pressure plasma (LTAPP) is an
electrical discharge produced by subjecting one or more
gases to electric field, either of constant or alternat- ing
amplitude (Moisan et al. 2001). It has been studied in
several fields such as polymerization (Uygun et al. 2011),
biological and chemical decontamination (Laroussi 2005).
Studies for dental purposes are relatively new and began
with dental-cavity disinfection (Sladek et al. 2007). There
are recent studies investigating its efficacy against end-
odontic micro-organisms in vitro (Jiang et al. 2009;
Yamazaki et al. 2011) or for root canal disinfection ex
vivo (Lu et al. 2009).
Ozone (O3) – a powerful oxidizing, water and nonpo- lar
solvent soluble, pale blue gas – exists in natural form in
atmosphere or it can be produced by generators (Braslavsky
and Rubin 2011). It has been used in the water industry,
disinfection of hospital rooms, dental unit water systems
(Dyas et al. 1983; Bocci 1992; Berrington and Pedler 1998).
Ozone therapy has been used for
wound-healing improvement, immune system modulation
and disinfection (Bocci and Di Paolo 2009), In dentistry,
its use has been recommended for soft tissue healing dur-
ing surgical procedures (Sandhaus 1969) and root caries
treatment (Baysan et al. 2000). In blood, ozone disinte-
grates forming reactive oxygen species and lipid oxidation
products that cause vasodilatation on the endothelium and
release of prostacyclin, interleukin-8, nitric oxide, platelet-
derived growth factors and transforming growth factor b,
which play a major role in rapid wound healing (Bocci
1999). Endodontic use of ozone is based on the early studies
of Zbinden (1951) and Overdiek and Hon- rath (1951), and
the research on ozone systems continues to develop new
applications and designs for use in root canals (Hems et al.
2005; Estrela et al. 2007; Case et al. 2012).
The aim of the present study was to compare the anti-
microbial efficacy of a new LTAPP design and a gaseous
ozone delivery system with 2·5% NaOCl on root canal walls
and dentine tubules in ex vivo root canal models infected
with Ent. faecalis.
Results and discussion
Ent. faecalis was chosen as the test micro-organism as it is
physically and ecologically strong (Ma et al. 2011),
concerning its prolonged survival capacity. The virulence
factors related to endodontic infection and the periradicu-
lar inflammatory response were listed as aggregation sub-
stance, surface adhesins, sex pheromones, lipoteichoic acid,
extracellular superoxide production, the lytic enzymes
gelatinase and hyaluronidase, and the toxin cytolysin
(Kayaoglu & Ørstavik 2004). It has the ability to
Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology
€ reyen Kaya et al.
B. U
SEM HV: 15·00 kV SEM MAG: 6·42 kx
Date(m/d/y): 04/07/11 Det: BSE Detector 10 µm VEGA\\ TESCAN
View field: 33·78 µm SEM Digital Microscopy Imaging
Plus Plazma Uygulama Sanay
SDU TEKNOKENT
Figure 1 A scanning electron microscopic view of dense Enterococ-
cus faecalis plaque (6·420 X). Cellular aggregation and colony forma-
tion on dentin surface can be seen following an incubation time of 4
weeks. White particles are dentin particles occurred during fractur-
ing by chisel.
form biofilm and survive as a monoculture (Stuart et al.
2006). Furthermore, it is spherical, and relatively small
cell diameter makes it easier to diffuse into dentinal
tubules. A three-week period of contamination allows dif-
fusion of the Ent. faecalis suspension through the dentine
tubules up to 300–400 lm, which slowly increases by time
(Haapasalo and Orstavik 1987). In SEM evaluations, we
detected a dense bacterial plaque after 4-week incuba- tion
(Fig. 1) similar to the biofilm micrographs of previ- ous
studies (Takemura et al. 2004; Case et al. 2012). Although
the observation of polysaccharide structure around the
clusters of micro-organisms proves the forma- tion of
biofilm in SEM and FESEM micrographs, in many
studies, SEM micrographs of biofilm were similar to our
micrograph in Fig. 1.
The degree of Ent. faecalis colonization (log10 CFU
values) did not show any significant difference among the
groups (P > 0·05). Significant differences were detected
among the groups for both microbial sample collection
methods after the disinfection procedures (P < 0·05) (Table
1). Bonferroni t (Dunn’s test)-test demonstrated that the
median log10 CFU value for the negative-control group
(saline) was significantly higher than that of the other
groups when paper points as well as dentinal sampling in
each third were used (P < 0·05). The median
9
 € reyen Kaya et al.
B. U Efficacy of Ozone and LTAPP on Ent. faecalis

Table 1 Mean and median log10 colony-forming units, standart deviations (SD), ranges and the number of the samples that showed no bacterial
growth (nNG)*

Microbial sampling of the root canals Groups n nNG Mean SD Median Range
c
With paper points LTAPP 12 5 2·47 2·29 2·95 0·00–5·47
d
NaOCl (Positive control) 12 11 0·50 0·66 0·00 0·00–1·90
b
Ozone 12 0 4·70 0·32 4·77 4·00–5·00
Saline (negative control) 6 0 5·66 0·61 5·50a 5·00–6·69
z
With dentinal sampling in coronal third LTAPP 12 4 3·34 2·47 4·87 0·00–5·30
z
NaOCl (Positive control) 12 6 2·34 2·45 2·25 0·00–5·30
y
Ozone 12 2 4·69 2·28 5·45 0·00–6·60
Saline (negative control) 6 0 x
6·54 0·26 6·47 6·30–7·00
b
With dentinal sampling in middle third LTAPP 12 12 0·00 0·00 0·00 0·00–0·00
a
NaOCl (positive control) 12 7 2·03 2·52 0·00 0·00–5·60
#
Ozone 12 2 4·72 2·29 5·66 0·00–6·47
Saline (negative control) 6 0 6·50 0·61 6·69* 5·30–6·95
ι
With dentinal sampling in apical third LTAPP 12 5 2·97 2·63 4·94 0·00–5·60
h,ι
NaOCl (Positive control) 12 5 3·19 2·82 5·31 0·00–6·00
h
Ozone 12 2 4·69 2·26 5·23 0·00–6·47
g
Saline (negative control) 6 0 6·38 0·61 6·65 5·30–6·84

*Superscript different letters indicate significances between the groups.

Table 2 Friedman followed Bonferroni t (Dunn’s test) test results of the groups for each root canal thirds*
Groups

NaOCl Saline
LTAPP (positive control) Ozone (negative control)

Root canal
thirds Mean (SD) Median Range Mean (SD) Median Range Mean (SD) Median Range Mean (SD) Median Range
Coronal 3·34 (2·47) 4·87ab 0–5·3 2·34 (2·46) 2·26y 0–5·3 4·69 (2·28) 5·45* 0–6·6 6·54 (0·27) 6·48a 6·3–7·0 Middle
0·00 0·00b 0–0 2·03 (2·52) 0·00x,y 0–5·6 4·72 (2·29) 5·66* 0–6·48 6·51 (0·61) 6·7a 5·3–6·9
Apical 2·97 (2·63) 4·94a 0–5·6 3·19 (2·82) 5·31x 0–6·0 4·69 (2·26) 5·24* 0–6·47 6·38 (0·61) 6·65a 5·3–6·8

*Superscript different letters indicate significances among the root canal thirds for each group.

log10 CFU value for the positive-control (NaOCl) group was
significantly lower than that of the other groups when
paper points were used (P < 0·05). While the mean log10
CFU value of LTAPP was less than that of the positive-
control group in the middle root canal third (P < 0·05),
there was no significant difference between them in coronal
and apical thirds (P > 0·05). Positive- control group was
more effective than ozone group in coronal and middle thirds
(P < 0·05), whereas they had similar median log10 CFU
values in apical third (P > 0·05) (Table 1).
Wilcoxon signed-rank test demonstrated that the CFU
value difference for all disinfection methods even in nega-
tive-control group obtained with paper points before and
after the disinfection procedures was statistically signifi-
cant (P < 0·05). The mean log10 CFU values of all groups
obtained after the disinfection procedures were less than
those obtained before (P < 0·05).
While the methods used in the present study did not
allow assessing biofilm removal, they allowed demonstrating
the disinfection efficacy on root canal walls and depth of
dentine tubules. The ex vivo model used in the present study
seems to be the most realistic model, as samples from dentin
chips enable the assessment of antibacterial activity in
dentinal tubules and accessory canals at differ- ent levels.
Peters et al. (2001) confirmed that grinding and culturing
gave better quantitative information about the extent of the
infection. Morphological variations of teeth can account for
differences between the samples. For a valid demonstration
of differences, at least one logarithmic step decrease in CFUs
is necessary (Ma et al. 2011).
Friedman test showed that there were significant differ-
ences among the root thirds in LTAPP (P = 0·003) and
positive-control (NaOCl) (P = 0·026) groups (Table 2). The
mean log10 CFU values obtained in apical thirds were higher
than those of coronal and middle thirds except for negative-
control (saline) group.
The number of samples with no bacterial growth (nNG)
observed after ozone application was significantly

10 Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology
 Efficacy of Ozone and LTAPP on Ent. faecalis
less than that of LTAPP and NaOCl applications (P
< 0·05), while there was no significant difference between
LTAPP and NaOCl applications (P > 0·05) (Table 1).
Various application times for the test groups can be
designed in such experiments. We decided to choose the
application time as 2 min for ozone according to manu-
facturer and a previous report (Case et al. 2012) and 5
min for LTAPP according to our preliminary study (Ureyen
Kaya et al. 2011). A recent study reported that 5, 10, 15 min
applications were effective on Ent. faecalis bio- films, and as
the exposure time increased, viable bacteria were
significantly reduced (Du et al. 2012).
As a positive control, we used 2·5% NaOCl similar with
the previous studies (Spratt et al. 2001; Hems et al. 2005).
The data of inhibitory concentration of NaOCl for Ent.
faecalis are not clear enough in the literature. Although
NaOCl 2·5% was reported to be effective in completely
eliminating Ent. faecalis in 10 min (Vianna et al. 2004;
Kustarci et al. 2009), in the present study a significant effect
was achieved with 2·5% NaOCl irriga- tion in only 2 min.
The bactericidal efficacy of ozone is based on forming
oxidated radicals in aqueous solutions, as a result of which
the cell membranes get damaged by altering the osmotic
stability and permeability (Dyas et al. 1983; Azarpazhooh
and Limeback 2008). However, there is no consensus on
application manner, time and optimum dosages of ozone to
achieve significant results. It has been reported by Hems et
al. (2005) that as the physical nature of sparging contributes
to the antibacterial effect of ozone, it should be delivered
under pressure for the pene- tration to the biofilm. However,
in the present study, ozone-enriched air bubbled into the
saline did not expose an intended antimicrobial efficacy as
NaOCl and LTAPP (Table 1). In addition, the present study
showed that antibacterial efficacy of ozone seems to be
superficial, because when the samples were collected with
dentin chips on coronal, middle and apical thirds, the log 10
med- ian CFU values were greater than those of samples col-
lected with paper points. Comparably, Case et al. (2012)
reported that ozone-enriched air for a total period of 2
min did not result in a reduction (71·6%) of the viable CFUs
as those (93·5%) in NaOCl group. Nagayoshi et al. (2004)
concluded that ozonated water had nearly the same
antimicrobial activity as 2·5% NaOCl during irriga- tion
especially when combined with sonication.
Similar to our findings, better performance of NaOCl over
gaseous ozone has been reported in previous studies in
eliminating micro-organisms in planktonic (Hems et al.
2005), or biofilm state (Hems et al. 2005; Case et al. 2012).
This low performance of gaseous ozone may be explained
with the claim of Restaino et al. (1995) that
Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology
€ reyen Kaya et al.
B. U
organic ingredient in the culture media (BHI) shields the
bacteria from the ozone through a redox reaction with
reductant in the media instead of the bacterial strain (Lynch
2009).
The production of short-lived chemical species in the gas
phase accounts for the antibacterial efficacy of LTAPP.
LTAPP destroys micro-organisms by disrupting the cell wall
using highly reactive free radicals, without the use of heat,
chemicals or pressure (Laroussi 1996). Under the conditions
of the present study, LTAPP had comparable disinfection
capacity with 2·5% NaOCl. It also seems to be effective in
deep parts of dentine. This was shown with the low-mean
log10 CFU values of dentine chip samples similar to those of
NaOCl and in contrast to those of ozone. Interestingly, the
antimicrobial effect of LTAPP at coronal third was lower
than that of the middle. This can be explained with the fact
that highest concentration of plasma energy is occurring 5–6
mm beyond the plasma needle (Kim et al. 2010). The plasma
needle produces bactericidal agents locally and no excess
radicals remain at the end of the treatment. LTAPP is
reported to have some advantages in biomedical
applications: low tempera- ture due to the heavy particles
(neutrals and ions) much lower than the temperature of the
electrons, which neither cause pain or bulk destruction of
living tissues (Stoffels 2002) nor damage heat-sensitive
materials (Philip et al. 2000).
It should be noted, however, that none of the treat-
ment regimens were able to render the canals free of bac-
teria in all samples. This proves the difficulty in dealing with
bacteria in root canals. The inability of all disinfec- tion
methods to completely kill the bacteria can be attrib- uted to
biofilm state of the micro-organism and deep penetration of
bacteria into the dentinal tubules. Gram- specific cell wall
structure of Ent. faecalis and complex anatomy of root canal
system are the other important factors (Kasahara et al.
1990). Three main components
that make up Ent. faecalis’s cell wall are peptidoglycan,
teichoic acid and polysaccharide. 40% of the cell wall is
made up of peptidoglycan, while the rest of the cell wall is
made up of a ‘rhamnose-containing polysaccharide and a
ribitol-containing teichoic acid’. The peptidoglycan resists
cytoplasmic osmotic pressure. Ent. faecalis is gener- ally
considered a nonencapsulated organism, shown by the
‘lack of a detectable mucoid phenotype’ (de la Maza et al.
2004).
In conclusion, the microbial sampling with paper points
through the root canal showed antibacterial effi- cacy of
NaOCl, LTAPP, ozone and saline in descending order,
respectively (P < 0·05). Dentinal sampling method allowed
the analysis of the efficacy of disinfectants in penetration
depth and killing of bacteria in tubules. No growth in all
samples of the middle third following
11
 € reyen Kaya et al.
B. U
LTAPP application was noticeable. Therefore, LTAPP was
superior to NaOCl in the middle third while both had similar
effects in coronal and apical thirds. LTAPP was more
effective than ozone in eliminating Ent. faecalis according
to both sampling methods. These findings led us to suggest
that LTAPP may be of great aid in end- odontic treatment
(Laroussi 2009). In the present study, no thermal effect or
stress on the surface of tooth was detected during the
experiment.
Nevertheless, further studies are necessary to determine
the optimum use in terms of clinical application manner and
time. The possible effect of LTAPP on biofilm should also
be investigated in in vivo situations.
Material and methods
Forty-two human mandibular premolars with straight root
canals extracted for periodontal reasons were selected with
simple random sampling from the teeth that were
anatomically in similar dimensions, fully developed apices
and free of cracks, caries or fractures (Fig. 2). Teeth were
decoronated to obtain roots 14 mm in length. Canal patency
was determined by passing a file (size 10 Kfile;
14
mm
(a) (b) (c)
(g) (h) (i) (j)
Figure 2 Schematic drawing of experimental design: (a) Selection of single rooted mandibular premolars (n = 46), (b) decoronation of teeth, (c)
root canal preparation (MAF: 40/.06), (d) autoclave sterilization, (e) contamination with Enterococcus faecalis, (f) SEM evaluation (n = 4), (g) nega-
tive control with saline (n = 6), (h) positive control with 2·5% NaOCl (n = 12), (i) ozone application (n = 12), (j) LTAPP application, (n = 12) (k)
12
Efficacy of Ozone and LTAPP on Ent. faecalis
Mani Co, Tokyo, Japan) through the apical foramen. The
root canals were prepared with crown-down technique by
using 0·06 taper ProFile (Dentsply Maillefer, Ballaigues,
Switzerland) to a size 40 master apical rotary instrument.
Irrigation was performed using 3 ml of 2·5% w/v NaOCl
after every change of instrument. A lubricant was used
(Glyde File Prep.; Dentsply Maillefer) throughout the
cleaning and shaping of the root canal. Smear layer was
removed by the sequential use of 3 ml of NaOCl, 17%
EDTA for 1 min, followed by distilled water for 1 min.
After root canal shaping, middle and apical thirds of
each sample were grooved by diamond burs. Root api- ces
were then sealed with nail varnish. Each root was autoclaved
at 121°C for 15 min. After sterilization, roots were
incubated in brain–heart infusion (BHI) broth for 48 h
at 37°C to ensure that there is no bacterial contamination.
Contamination with Ent. faecalis
A liquid culture suspension of 1 McFarland standard BHI
broth in pH 7 ·2 was prepared to obtain 3 9 108 colony-
forming units per mL (CFU ml —1) from a subculture of
Autoclave
steam
E. faecalis
121°C
(d) (e) (f)
(k) (l) (m) (n)
Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology
 microbial sampling with sterile paper points (n = 42), (l) horizontal sectioning in three levels, (m) vertical sectioning (n) and dentin chips collection.
Efficacy of Ozone and LTAPP on Ent. faecalis € reyen Kaya et al.
B. U

Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology 13
 € reyen Kaya et al.
B. U Efficacy of Ozone and LTAPP on Ent. faecalis

Ent. faecalis (ATCC 29212). The sterilized tooth was placed
in an Eppendorf tube, (2 ml) of the bacterial sus- pension
was added into this tube and it was incubated for 4 weeks
under aerobic and static conditions at 37°C. The medium
was changed every 2 days to avoid satura- tion and
confirmed the growth of Ent. faecalis, and the cultures were
checked for purity by Gram stain and colony morphology on
BHI agar with 10% sheep blood. Four ran- domly selected
samples were examined to confirm the presence of dense
Ent. faecalis plaque by scanning electron microscopy
(SEM).
Experimental design
After the incubation period, roots were assigned randomly
to 1 of 4 groups. Root canals were irrigated using 27-gauge
dental injector placed 1 mm far from the working length for
2 min in negative and positive-control groups. In nega- tive-
control group (n = 6), 5 ml sterile saline and in posi-
tive-control group (n = 12) 5 ml 2 ·5% NaOCl were used.
In the ozone group (n = 12), a dental ozone system (Prozone;
W&H Dental Werk Burmoos GmbH, Burmoos, Austria)
was used by attaching a sterile endodontic can- nula. Root
canals were filled with 100 ll sterile saline solution, and
then, sterile cannula was inserted to the root canal until 2 mm
short of the working length. Ozone- enriched air (140 ppm,
2 l min—1) was delivered for 24 s as recommended by the
manufacturer. The cannula was removed from the canal
after each 24-s cycle to prevent
room air being delivered during system purging. The canal
was then refilled with fresh saline, and the ozone treatment
was repeated four times giving a total ozonation time of 2
min. In LTAPP group (n = 12), an experimental device made
of dental syringe was used for guiding the gas flow. Plasma
jet was obtained by designing a dielectric barrier discharge.
The voltage-current characteristic of sinusoidal driven
source was a peak-to-peak voltage of 16 kV at an excitation
frequency of 10 kHz. The outside diameter of the needle was
1 mm and inside diameter 0·5 mm with a length of 13 mm.
Thickness of the dielectric barrier mate- rial was 6 mm.
LTAPP was applied for 5 min through the needle that was
inserted 1 mm into the root, and the gas flow through the root
canal was observed. The oxygen and helium flow rates were
0·2 lpm and 5 lpm, respectively. The schematic of the
experimental set-up is given in Fig. 3.
Microbial sampling of the canals
All procedures were carried out in a laminar flow cham- ber
using sterile instruments. Root canals were sampled before
and after treatment protocols using sterile paper points
placed for 60 s. Following to each sampling, paper points
were transferred to tubes containing 1 ml of freshly
prepared BHI broth and vortexed for 1 min. After 10-fold
serial dilutions, aliquots of 0·1 ml were plated onto BHI
agar plates with the aid of a Drigalsky spatula and incubated
at 37°C for 24 h. The colony-forming units (CFUs) grown
were counted and recorded.

Oscilloscope

Plasma pencil Flowmeter

Voltage Current H.V Electrode

monitor monitor Plasma

Tooth

High voltage
power supply

H.V He O2
Output

Ground
Dielectric barrier

Figure 3 The schematic of the experimental set-up of LTAPP. The voltage-current characteristic of sinusoidal driven source was a peak-to-peak
voltage of 16 kV at an excitation frequency of 10 kHz. The outside diameter of the needle was 1 mm and inside diameter 0·5 mm with a length
of 13 mm. Thickness of the dielectric barrier material was 6 mm. LTAPP was applied for 5 min through the needle that was inserted 1 mm into
the root, and the gas flow through the root canal was observed. The oxygen and helium flow rates were 0·2 lpm and 5 lpm, respectively.

14 Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology
 Efficacy of Ozone and LTAPP on Ent. faecalis
Dentinal sampling
Each sample was fractured into coronal, middle and api- cal
sections by using a sterile chisel. For evaluation of the
antimicrobial effectiveness of treatment protocols, dentin
chips were taken from each section of root canal wall
surface. A low-speed endodontic handpiece (X-Smart,
Dentsply Maillefer, Ballaigues, Switzerland) with an ISO
012 tungsten carbide round, bur were used for collecting
dentin chips from root sections. The dentin chips samples
and each bur collected in each third were immediately
placed into separate sterile Eppendorf tubes containing 1
ml BHI broth. In order to standardize the removed volume
of the dentine chips, the Eppendorf tubes were weighed
before and after dentine collection until 10 mg dentin chips
were obtained.
Growing colonies were counted and recorded as CFUs 24
h later and converted to their log10 values. All assays were
repeated three times, and the purity of positive cultures was
confirmed by Gram staining, catalase production, colony
morphology on BHI blood agar.
Statistical analysis
Box’s M test showed that the data did not provide pre-
conditions of homogeneity of variance (P < 0·05) and
were not normally distributed according to Kolmogorov–
Smirnov Test (P < 0·05). Thus, the significance among
the groups was statistically analysed with Kruskal–Wallis
test followed by Bonferroni t (Dunn’s test) (P = 0·05).
The statistical difference between the CFU values obtained
with paper points after the contamination with Ent.
faecalis and disinfection procedures was analysed with
Wilcoxon signed-rank test for each group (P = 0·05). The
CFU values obtained from dentinal sampling of each root
canal third were compared using Friedman test followed
by Bonferroni t (Dunn’s test) (P = 0·05).
The number of the samples that showed no bacterial
growth (nNG) in positive control, ozone, LTAPP and
negative-control groups was statistically analysed with z-
test (P = 0·05).
Conflict of interest
No conflict of interest declared.
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