ORI GI NAL ARTI CLE

Efficacy of endodontic applications of ozone and low-
temperature atmospheric pressure plasma on root canals
infected with Enterococcus faecalis
B.

Ureyen Kaya
1
, A.D. Kececi
1
, H.E. G€ uldas ß
1
, E.S. Cßetin
2
, T.

Ozt€ urk
2
, L.

Oksuz
3
and F. Bozduman
3
1 Department of Endodontics, Faculty of Dentistry, S€ uleyman Demirel University, Isparta, Turkey
2 Department of Microbiology, Faculty of Medicine, S€ uleyman Demirel University, Isparta, Turkey
3 Department of Physics, Faculty of Science & Arts, S€ uleyman 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

Ureyen Kaya, S€ uleyman Demirel

Univ-
ersitesi, Dis ß Hekimli gi Fak€ ultesi, Endodonti
AD, 32260 Kamp€ us-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 2Á5% 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 = 0Á05). The microbial sampling with paper
points showed antibacterial efficacy of NaOCl, LTAPP, ozone and saline in
descending order, respectively (P < 0Á05). The microbial sampling with dentin
chips demonstrated a superior efficacy of LTAPP compared with NaOCl in the
middle third (P < 0Á05), while both had similar effects in coronal and apical
thirds (P > 0Á05). NaOCl and LTAPP were better than ozone at the coronal
and middle parts of the root canals (P < 0Á05). These findings led us to
suggest that LTAPP, which has no thermal and chemical effects, may be of
great aid in endodontic treatment.
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
canal infections in its planktonic or biofilm forms
(George 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
Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology 8
Letters in Applied Microbiology ISSN 0266-8254
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 2Á5% 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
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 (log
10
CFU
values) did not show any significant difference among the
groups (P > 0Á05). Significant differences were detected
among the groups for both microbial sample collection
methods after the disinfection procedures (P < 0Á05)
(Table 1). Bonferroni t (Dunn’s test)-test demonstrated
that the median log
10
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 < 0Á05). The median
SEM HV: 15·00 kV SEM MAG: 6·42 kx
10 µm Date(m/d/y): 04/07/11 Det: BSE Detector
SEM View field: 33·78 µm
VEGA\ TESCAN
Digital Microscopy Imaging
Plus Plazma Uygulama Sanay
SDU TEKNOKENT
Figure 1 A scanning electron microscopic view of dense Enterococ-
cus faecalis plaque (6Á420 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.
Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology 9
B.

Ureyen Kaya et al. Efficacy of Ozone and LTAPP on Ent. faecalis
log10 CFU value for the positive-control (NaOCl) group
was significantly lower than that of the other groups
when paper points were used (P < 0Á05). While the mean
log
10
CFU value of LTAPP was less than that of the
positive-control group in the middle root canal third
(P < 0Á05), there was no significant difference between
them in coronal and apical thirds (P > 0Á05). Positive-
control group was more effective than ozone group in
coronal and middle thirds (P < 0Á05), whereas they had
similar median log10 CFU values in apical third
(P > 0Á05) (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 < 0Á05). The mean log
10
CFU values of all groups
obtained after the disinfection procedures were less than
those obtained before (P < 0Á05).
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 = 0Á003) and
positive-control (NaOCl) (P = 0Á026) groups (Table 2).
The mean log
10
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
Table 1 Mean and median log
10
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
With paper points LTAPP 12 5 2Á47 2Á29 2Á95
c
0Á00–5Á47
NaOCl (Positive control) 12 11 0Á50 0Á66 0Á00
d
0Á00–1Á90
Ozone 12 0 4Á70 0Á32 4Á77
b
4Á00–5Á00
Saline (negative control) 6 0 5Á66 0Á61 5Á50
a
5Á00–6Á69
With dentinal sampling in coronal third LTAPP 12 4 3Á34 2Á47 4Á87
z
0Á00–5Á30
NaOCl (Positive control) 12 6 2Á34 2Á45 2Á25
z
0Á00–5Á30
Ozone 12 2 4Á69 2Á28 5Á45
y
0Á00–6Á60
Saline (negative control) 6 0 6Á54 0Á26 6Á47
x
6Á30–7Á00
With dentinal sampling in middle third LTAPP 12 12 0Á00 0Á00 0Á00
b
0Á00–0Á00
NaOCl (positive control) 12 7 2Á03 2Á52 0Á00
a
0Á00–5Á60
Ozone 12 2 4Á72 2Á29 5Á66
#
0Á00–6Á47
Saline (negative control) 6 0 6Á50 0Á61 6Á69* 5Á30–6Á95
With dentinal sampling in apical third LTAPP 12 5 2Á97 2Á63 4Á94
ι
0Á00–5Á60
NaOCl (Positive control) 12 5 3Á19 2Á82 5Á31
h,ι
0Á00–6Á00
Ozone 12 2 4Á69 2Á26 5Á23
h
0Á00–6Á47
Saline (negative control) 6 0 6Á38 0Á61 6Á65
g
5Á30–6Á84
*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*
Root canal
thirds
Groups
LTAPP
NaOCl
(positive control) Ozone
Saline
(negative control)
Mean (SD) Median Range Mean (SD) Median Range Mean (SD) Median Range Mean (SD) Median Range
Coronal 3Á34 (2Á47) 4Á87
ab
0–5Á3 2Á34 (2Á46) 2Á26
y
0–5Á3 4Á69 (2Á28) 5Á45* 0–6Á6 6Á54 (0Á27) 6Á48
a
6Á3–7Á0
Middle 0Á00 0Á00
b
0–0 2Á03 (2Á52) 0Á00
x,y
0–5Á6 4Á72 (2Á29) 5Á66* 0–6Á48 6Á51 (0Á61) 6Á7
a
5Á3–6Á9
Apical 2Á97 (2Á63) 4Á94
a
0–5Á6 3Á19 (2Á82) 5Á31
x
0–6Á0 4Á69 (2Á26) 5Á24* 0–6Á47 6Á38 (0Á61) 6Á65
a
5Á3–6Á8
*Superscript different letters indicate significances among the root canal thirds for each group.
10 Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology
Efficacy of Ozone and LTAPP on Ent. faecalis B.

Ureyen Kaya et al.
less than that of LTAPP and NaOCl applications
(P < 0Á05), while there was no significant difference
between LTAPP and NaOCl applications (P > 0Á05)
(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 2Á5% 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 2Á5% 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 2Á5% 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 (71Á6%) of the viable
CFUs as those (93Á5%) in NaOCl group. Nagayoshi et al.
(2004) concluded that ozonated water had nearly the
same antimicrobial activity as 2Á5% 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
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 2Á5% NaOCl. It also seems to
be effective in deep parts of dentine. This was shown with
the low-mean log
10
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 < 0Á05). 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
Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology 11
B.

Ureyen Kaya et al. Efficacy of Ozone and LTAPP on Ent. faecalis
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;
Mani Co, Tokyo, Japan) through the apical foramen. The
root canals were prepared with crown-down technique by
using 0Á06 taper ProFile (Dentsply Maillefer, Ballaigues,
Switzerland) to a size 40 master apical rotary instrument.
Irrigation was performed using 3 ml of 2Á5% 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 7Á2 was prepared to obtain 3 9 10
8
colony-
forming units per mL (CFU ml
À1
) from a subculture of
(a) (b)
14
mm
Autoclave
steam
121°C
E
.

f
a
e
c
a
l
i
s
S
a
l
i
n
e
N
a
O
C
I
O
z
o
n
e
L
T
A
P
P
(d) (e) (f) (c)
(g) (h) (j) (k) (l) (m) (n) (i)
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 2Á5% NaOCl (n = 12), (i) ozone application (n = 12), (j) LTAPP application, (n = 12) (k)
microbial sampling with sterile paper points (n = 42), (l) horizontal sectioning in three levels, (m) vertical sectioning (n) and dentin chips collection.
12 Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology
Efficacy of Ozone and LTAPP on Ent. faecalis B.

Ureyen Kaya et al.
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 2Á5% 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
À1
) 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 0Á5 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 0Á2 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 0Á1 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
Voltage
monitor
Current
monitor
High voltage
power supply
H.V
Output
H.V Electrode
Flowmeter
Plasma
Tooth
Ground
Dielectric barrier
He O
2
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 0Á5 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 0Á2 lpm and 5 lpm, respectively.
Letters in Applied Microbiology 58, 8--15 © 2013 The Society for Applied Microbiology 13
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Ureyen Kaya et al. 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 log
10
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 < 0Á05) and
were not normally distributed according to Kolmogorov–
Smirnov Test (P < 0Á05). Thus, the significance among
the groups was statistically analysed with Kruskal–Wallis
test followed by Bonferroni t (Dunn’s test) (P = 0Á05).
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 = 0Á05). 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 = 0Á05).
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 = 0Á05).
Conflict of interest
No conflict of interest declared.
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Ureyen Kaya et al. Efficacy of Ozone and LTAPP on Ent. faecalis