doi:10.1111/j.1365-2591.2011.01959.
Antimicrobial effect of endodontic solutions used
as final irrigants on a dentine biofilm model
R. Ordinola-Zapata1, C. M. Bramante1, B. Cavenago1, M. S. Z. Graeff2, I. Gomes de Moraes1,
M. Marciano1 & M. A. H. Duarte1
1
Department of Endodontics, Dental School of Bauru, University of São Paulo, Bauru; and 2Integrated Research Center (CIP),
Dental School of Bauru, University of São Paulo, Bauru, Brazil
Abstract
Ordinola-Zapata R, Bramante CM, Cavenago B, Graeff
MSZ, Gomes de Moraes I, Marciano M, Duarte MAH.
Antimicrobial effect of endodontic solutions used as final
irrigants on a dentine biofilm model. International Endodontic
Journal, 45, 162–168, 2012.
Aim To evaluate the residual biovolume of live
bacterial cells, the mean biofilm thickness and the
substratum coverage found in mixed biofilms treated
with different endodontic irrigant solutions.
Methodology Twenty-five bovine dentine specimens
were infected intraorally using a removable orthodontic
device. Five samples were used for each irrigant solution:
2% chlorhexidine, 1% sodium hypochlorite (NaOCl),
10% citric acid, 17% EDTA and distilled water. The
solutions were used for 5 min. The samples were stained
using the Live/Dead technique and evaluated using a
confocal microscope. Differences in the amount of total
biovolume (lm3), number of surviving cells (lm3), mean
Introduction
Biofilms can be defined as bacterial cells attached to a
surface embedded in an exopolysaccharide matrix that
fills the space between the cells (Costerton 2007).
Biofilms are present in necrotic pulp canal spaces of
primary and secondary root canal infections (Ricucci
et al. 2009). A previous study (Ricucci & Siqueira
2010) has reported that apical periodontitis meets the
Correspondence: Ronald Ordinola-Zapata, Faculdade de
Odontologia de Bauru, USP, Octávio Pinheiro Brisolla, 9-75,
CEP 17012-901, Bauru, São Paulo, Brazil (Tel.: +55-
1432358344; fax: +55-1432358344; e-mail: ronaldordin
ola@usp.br).
162 International Endodontic Journal, 45, 162–168, 2012
biofilm thickness (lm) and substratum coverage (%) of
the treated biofilms were determined using nonpara-
metric statistical tests (P < 0.05).
Results Similar values of biovolume total, biovolume
of live subpopulations and substratum coverage were
found in 2% chlorhexidine, 10% citric acid, 17% EDTA
and distilled water-treated biofilms (P > 0.05). The
lower values of the studied parameters were found in
1% NaOCl-treated dentine (P < 0.05) with the excep-
tion of the mean biofilm height criteria that did not
reveal significant differences amongst the irrigant
solutions (P > 0.05).
Conclusions One per cent sodium hypochlorite was
the only irrigant that had a significant effect on biofilm
viability and architecture.
Keywords: confocal laser scanning microscopy,
dentin, irrigant solutions, oral biofilms.
Received 28 April 2011; accepted 28 August 2011
criteria proposed by Parsek & Singh (2003) to define a
biofilm-associated disease as: bacteria associated to a
substratum, bacteria encased in a matrix of bacterial or
host constituents, infection localized and resistant to
antibiotic therapy.
Although the eradication of intraradicular biofilms
should be ideally performed by mechanical methods,
the instrumentation process cannot directly access a
considerable amount of infected tissue owing to the
irregularities of the root canal system (Paque et al.
2009, 2010). For this reason, antimicrobial com-
pounds are used during instrumentation and as a final
rinse before canal filling. Several irrigant solutions have
been proposed for use during the final rinse. EDTA and
citric acid are used to eliminate the smear layer pro-
ª 2011 International Endodontic Journal
 Ordinola-Zapata et al. The effect of irrigants on a dentin biofilm model
duced by the action of endodontic instruments on root
canal walls (Violich & Chandler 2010, Prado et al.
2011). Others irrigants, such as 2% chlorhexidine,
have been proposed to enhance antimicrobial control at
the end of the chemo-mechanical process (Zamany
et al. 2003). Although EDTA and citric acid have been
reported to have no or only a slight antimicrobial effect
(Siqueira et al. 1998, Arias-Moliz et al. 2009), previous
reports have concluded that EDTA can remove 83% of
a Streptococcus gordonii biofilm (Chavez de Paz et al.
2010). Whilst a final rinse with an antimicrobial
compound is highly desirable, it is not clear how
beneficial it would be on residual biofilms that were
obviously not affected by the primary chemo-mechan-
ical treatment.
Dentine is a mineralized connective tissue that is
porous because of the presence of dentinal tubules.
Microorganisms are usually able to adhere to its surface
and to enter into root dentinal tubules (Love &
Jenkinson 2002). For this reason, infected dentine is
commonly found in primary and secondary root canal
infections (Ricucci et al. 2009). The use of dentine as a
substrate to induce microbial biofilms has been used
commonly to test the effect of endodontic procedures
mainly in combination with scanning electron micros-
copy or culture techniques (George et al. 2005, Clegg
et al. 2006, Bhuva et al. 2010). However, SEM meth-
ods do not provide appropriate three-dimensional
quantification of the residual biomass, and culture
techniques depend on a representative sample that is
taken after dispersing the microbial cells (Costerton
2007). Direct comparisons addressing three-dimen-
sional quantification of the affected biomass attached
to dentine after exposure to common irrigants solutions
are unknown.
Direct observation techniques using confocal micros-
copy can provide data on bacterial quantification
(Lawrence et al. 1991, Costerton 2007). Previous
research has shown that in situ microscopic quantifi-
cation of the live bacterial biomass is better represented
in comparison with UFC quantification using culture
techniques (Shen et al. 2010), and this is an important
task when the limits of antimicrobial compounds need
to be determined. Thus, this study aimed to evaluate
the residual biovolume of live cells, mean biofilm height
and the substratum coverage found in mixed biofilms
treated with irrigant solutions used commonly during
final rinse procedures such as: 2% chlorhexidine, 17%
EDTA, 10% citric acid and distilled water. The effect of
1% sodium hypochlorite was also tested for compara-
tive purposes. The null hypothesis was that: biofilms
ª 2011 International Endodontic Journal
treated with irrigant solutions used for final rinse
purposes do not result in different amounts of live cells,
substratum coverage and mean biofilm thickness.
Materials and methods
The irrigant solutions used were: 1% sodium hypo-
chlorite (CloroRio, São José do Rio Preto, Brazil), 10%
Citric Acid (Farmacia Especı́fica, Bauru, Brazil), 17%
EDTA (Biodinamica, Ibiporã, Brazil) and 2% chlorhex-
idine (Villevie, Joinville, Brazil). Distilled water was used
for control purposes.
Twenty-five dentine sections (3 · 3 · 2 mm) were
obtained from previously sterilized bovine root dentine.
The samples were treated with 1% sodium hypochlorite
(NaOCl) for 15 min and 17% EDTA for 3 min to
eliminate organic debris and the smear layer produced
during the sectioning process. Five dentine samples
were fixed in each experimental procedure on a Hawleys
orthodontic device to produce biofilm. To standardize
the rate of biofilm development, one healthy volunteer
used the intraoral device for 48 h except during regular
oral hygiene practices. The ethical human committee
approved the protocol (CEP-134/2010). After the
intraoral infection process, each sample was incubated
in 2 mL of brain heart infusion (BHI) at 37 C for 12 h
using 24-well tissue culture plates.
After the incubation period, the dentine samples
were washed with 1 mL of distilled water to eliminate
the culture medium and nonadherent cells. The contact
test was performed by immersing the dentine sample
for 5 min in 1 mL of the irrigant solutions tested.
Twenty-four-well tissue culture plates were used for the
contact test. One additional dentine sample was treated
with 1 mL distilled water for 5 min to serve as control.
Five samples were used for each irrigant solution.
After the contact test, the 1% NaOCl samples were
treated with 100 lL of 5% sodium thiosulfate and a
final rinse of distilled water was used. Chlorhexidine,
10% citric acid and 17% EDTA-treated samples were
washed with distilled water. A pilot study revealed that
sodium thiosulfate did not have a dissolution ability
and improved the quality of the staining process for
sodium hypochlorite-treated dentine.
After the contact test, the samples were stained using
the Syto-9/Propidium iodide technique (Live/Dead,
Bacligth; Invitrogen, Eugene, OR, USA); SYTO-9 is a
green fluorescent nucleic acid stain, generally labelling
both live and dead microorganisms. PI is a red fluo-
rescent nucleic acid stain and penetrates only the cells
with damaged membranes, thus visualizing dead
International Endodontic Journal, 45, 162–168, 2012 163
 The effect of irrigants on a dentin biofilm model Ordinola-Zapata et al.
microbes (Shen et al. 2009). The concentration of the
fluorochromes was adjusted during a pilot study. To
improve the contrast between the biofilm cells and
dentine, 1 lL of each dye was added to 1 mL of distilled
water. From this working solution, 200 lL was added
to the treated dentine samples and controls for 20 min
at room temperature in a dark environment. This dye
concentration avoids the staining of dentine allowing
an appropriate quantification using specially dedicated
biofilm software.
All the samples were observed using a confocal laser
scanning microscope (CLSM Leica TCS-SPE; Microsys-
tems GmbH, Mannheim, Germany). Four confocal
pictures were obtained from each sample using the
40X oil lens and a 1 lm step size in a format of
512 · 512 pixels. The sequential frame scan mode was
used to prevent crosstalk. At least 7 lm of the scanning
process included the subsurface level of the dentine.
Each 40X biofilm picture represented an area of
275 · 275 lm2. For quantification purposes the bioi-
mage_L software (http://www.bioimagel.com) was
used, which produced information on the total biofilm
population as well as the independent subpopulations
represented by red and green fluorescent colours
(Chavez de Paz 2009). The biofilm analysis tool of the
software was used to evaluate the four stacks obtained
for each sample. The data obtained in the biovolume/
stack analysis of five independent experiments were
pooled as a single column to provide a single mean
representative of the 20 evaluated stacks in each
irrigant solution. The parameters evaluated were bio-
volume of total and green population (live cells) in lm3,
mean biofilm height in lm and substratum coverage
that was expressed in terms of percentage. The biovo-
lume is the volume occupied by microorganisms in a
3D space. The mean biofilm height calculates the mean
vertical expansion and the substratum coverage shows
how efficiently the organism colonizes the dentine
(Chavez de Paz et al. 2010). Representative confocal
Table 1 Median, mean and range values of total and green biovolume after the contact with the experimental solutions
1% NaOCl 17% EDTA
3
Biovolume total (lm )
Median 3.04 · 105 5.30 · 106
Mean 7.88 · 105 6.36 · 106
Range 1.60 · 103–4.35 · 106 1.02 · 106–1.79 · 107
Live cells (lm3)
Median 5.98 · 103 4.23 · 106
Mean 3.24 · 105 4.60 · 106
Range 0–2.30 · 106 3.84 · 105–1.20 · 107
164 International Endodontic Journal, 45, 162–168, 2012
pictures in the reflection mode were also taken to
observe the surfaces of dentine after treatment with the
irrigant solutions. Tridimensional models were recon-
structed using the OsiriX software (http://www.osirix-
viewer.com) running under Snow Leopard Mac OSX
10.5.8 software (Apple, Cupertino, CA, USA).
Preliminary analysis of the data did not show a
normal distribution. Thus, the Kruskal–Wallis and
Dunn tests were used for multiple comparisons
amongst the groups. The level of significance was set
at P < 0.05, and Prisma 5.0 (GraphPad Software Inc,
La Jolla, CA, USA) was used as the analytical tool.
Results
A total of 100 confocal ‘stacks’ were evaluated for all
the irrigant solution tested (20 for each irrigant
solution). In all the biofilms evaluated, the maximum
biofilm thickness ranged from 20–30 lm without
statistical significances amongst the groups (data not
shown). The median, mean and range values of
biovolume total, biovolume of the green sub-population
expressed in lm3 and substratum coverage (%) are
presented in Table 1 and Fig. 1. No statistical differ-
ences were found for these parameters tested when the
biofilms were treated with distilled water, 10% citric
acid, 17% EDTA or 2% chlorhexidine (P > 0.05). The
lower values of the studied parameters were found in
1% NaOCl-treated dentine (P < 0.05) with the excep-
tion of mean biofilm height that did not show statistical
differences amongst the irrigant solutions (P > 0.05).
(See Fig. 1). Chlorhexidine-treated biofilms showed a
discrete reduction of the viable cells in comparison with
the distilled water group but the difference was not
significant.
Although some clean dentine surfaces were observed
in the 1% NaOCl-treated specimens, residual biofilms
appeared firmly attached into the dentine structure.
Complete cleaning of dentine was not observed in any
10% Citric acid 2% Chlorhexidine Distilled water
5.39 · 106 7.26 · 106 5.42 · 106
6.37 · 106 7.33 · 106 7.68 · 106
1.23 · 106–1.36 · 107 7.11 · 105–1.68 · 107 2.72 · 106–1.64 · 107
4.44 · 106 2.14 · 106 4.69 · 106
4.87 · 106 2.76 · 106 6.67 · 106
2.53 · 105–1.20 · 107 1.92 · 104–7.71 · 106 1.69 · 106–1.52 · 107
ª 2011 International Endodontic Journal
 Ordinola-Zapata et al. The effect of irrigants on a dentin biofilm model
(a)
(b)
Figure 1 Box-plots of mean biofilm
height in lm (a) and substratum cover-
age values in terms of percentage (b)
after 5 min of contact with the irrigant
solutions.
of the tested solutions, neither in confocal fluorescence
or the reflection picture. In all samples an intense
bacterial colonization was observed at the entrance of
dentinal tubules. Representative pictures of the treated
biofilms are shown in Figs 2 and 3.
Discussion
The use of intraorally infected dentine and mixed
biofilm models formed from subgingival plaque have
been applied in previous studies to test the effect of
endodontic antimicrobial compounds as irrigants and
intracanal dressings (Barthel et al. 2002, Virtej et al.
2007, Shen et al. 2009). Because dead oral bacteria are
commonly an integral part of oral biofilms (Auschill
et al. 2001, Arweiler et al. 2004), the samples of this
study were additionally incubated for 12 h under rich
supplement conditions (BHI). Using this method at least
85% of the biomass was found to be viable when the
biofilm was treated with a nonantimicrobial compound
(water).
Despite the clear advantages of the biofilm model
used in the present study, it is difficult to reproduce the
real clinical conditions in which the antimicrobial
agent diffuses into the root canal system at the apical
level. The use of the present biofilm model can serve to
ª 2011 International Endodontic Journal
identify variables in which bacteria can survive appli-
cation of strong antibacterial compounds. One of these
variables appears to be the presence of anatomical
irregularities. The presence of a fissure or fin extending
into the dentine structure not only can increase the
vertical dimension in which the biomass can grow but
also can limit the antimicrobial action (Deng & ten Cate
2004). Other interesting variables to be tested are if the
surviving cells or new colonizers can attach to the
residual biofilm or if ultrasonic activation has an
additive effect on biofilm dissolution (Harrison et al.
2010). Although a detailed description of the oral
bacterial species that survive antimicrobial stress is
beyond the scope of this study, future research is
needed on this topic.
The use of dentine as a substrate allows the
identification of the presence of bacteria cells several
micrometres (7 lm) above the surface. However, the
detection of viable bacteria in the deep layers of the
dentine remains restricted by the limited penetration of
the laser during the scanning process. Retamozo et al.
(2010) showed that 40–60 min of 5.25% NaOCl are
necessary to obtain a negative culture in infected
dentine and thus confirmed the challenge of eliminat-
ing infection in the deep dentine layers. Future
improvements of the CLSM experimental method may
International Endodontic Journal, 45, 162–168, 2012 165
 The effect of irrigants on a dentin biofilm model Ordinola-Zapata et al.
(a) (b)
(c) (d)
(e) (f)
(g) (h)
address the viability of bacteria inside dentinal tubules
more precisely (Zapata et al. 2008).
The significantly lower values of substratum cover-
age found in 1% NaOCl-treated biofilms may be
explained by the dissolution effect that decreased the
biovolume in the horizontal dimension. However, the
mean biofilm height was not significantly different
amongst the irrigant solutions. This results show that
complete biofilm dissolution in both vertical and
166 International Endodontic Journal, 45, 162–168, 2012
Figure 2 Representative three-dimen-
sional constructions (left) and confocal
sections of dentine surfaces (right) after
the treatment with: distilled water (a–b),
17% EDTA (c–d), 10% citric acid (e–f)
and 2% chlorhexidine (g–h). Infection of
dentinal tubules is visible in all the
confocal sections. Bars in confocal sec-
tions represent 40 lm. Three-dimen-
sional constructions represent an area of
275 · 275 lm2.
horizontal dimensions is difficult using only 5 min
contact time. Longer decontaminating periods were
avoided in this study because EDTA, citric acid and 2%
chlorhexidine are generally used for short time periods
during root canal treatment.
Antimicrobial activity is desirable for all the irrigant
solutions used during root canal treatment including
chelating agents. However, the antimicrobial limits of
endodontic irrigants should be recognized. A contact
ª 2011 International Endodontic Journal
 Ordinola-Zapata et al. The effect of irrigants on a dentin biofilm model
(a)
(b)
(c)
Figure 3 The effect of 5 min of 1% NaOCl on biofilm structure
can be seen in (a) and (b). After 5 min, there is a partial
disruption of the biofilm structure and some viable cells
(green) are still covering the dentine (a). However, the
confocal reflection picture shows that biofilm layers are still
covering partially the dentine structure (c)
ª 2011 International Endodontic Journal
time of 5 min of 17% EDTA and 10% citric acid had no
effect on the biofilm viability results that are in
agreement with Arias-Moliz et al. (2009). The effect of
2% chlorhexidine on the biofilm viability was also
limited. Limited anti-biofilm activity of this compound
in comparison with NaOCl has been found in previous
studies (Clegg et al. 2006, Dunavant et al. 2006, Hope
et al. 2010, Chavez de Paz et al. 2010). Under clinical
conditions, the antimicrobial activity of 2% chlorhex-
idine could be expected only in well-cleaned root canal
walls; on the other hand, there may be no effect of this
compound on the architecture or viability on biofilms
situated in areas unaffected by the primary chemo-
mechanical process. In addition, the strong antimicro-
bial effect of 1% NaOCl on biofilms attached to dentine
was shown in this study confirming the results of
others (Bryce et al. 2009, Hope et al. 2010, Bhuva et al.
2010). Overall, 1% sodium hypochlorite-treated bio-
films removed 90% of the total biovolume and killed
more bacteria in comparison with the other irrigant
solutions. Thus, rejecting the null hypothesis.
Conclusion
One per cent sodium hypochlorite was the only irrigant
that showed a significant effect on the biofilm viability
and architecture.
Acknowledgements
This work was supported by FAPESP (2010/16002-4).
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