Basic Research—Technology
Residual and Antimicrobial Activity of Final Irrigation
Protocols on Enterococcus Faecalis Biofilm in Dentin
Pilar Baca, DDS, MD, PhD,* Pilar Junco, DDS, MD, PhD,* Mar
ıa Teresa Arias-Moliz, DDS, PhD,†
alez-Rodr
ıguez, DDS, PhD,* and Carmen Mar
ıa Ferrer-Luque, DDS, MD, PhD*
Mar
ıa Paloma Gonz

Abstract
Introduction: The use of root canal irrigating solutions
exerting antimicrobial activity and prolonged residual
activity is desirable in order to control dentin infection
and delay reinfection of the root canal. The aim of this
study was to evaluate the residual antimicrobial activity
and the capacity to eradicate Enterococcus faecalis
biofilm of different irrigating solutions, alone and in
combination, in a dentin-volumetric test. Methods:
Solutions of 2.5% sodium hypochlorite (NaOCl), 2%
chlorhexidine (CHX), 0.2% cetrimide (CTR), 17% ethyl-
endiaminetetraacetic (EDTA), 7% maleic acid (MA),
and regimens of 2.5% NaOCl followed by 17% EDTA
or 7% MA and 0.2% CTR or 2% CHX were used to deter-
mine their residual activity by exposing treated dentin
blocks to E. faecalis for 24 hours. Antimicrobial activity
was assayed on 3-week biofilm formed on dentin
blocks. Results of residual activity and antimicrobial
activity were respectively expressed as the inhibition
percentage of biofilm formation and the kill percentage
of biofilm. Results: A 2% CHX and 0.2% CTR solution
showed 100% biofilm inhibition; 2.5% NaOCl showed
the lowest residual activity (18.10%). The kill
percentage of 2.5% NaOCl and 0.2% CTR was 100%
followed by 7% MA and 2% CHX, whereas 17% EDTA
was the least effective (44%). Solutions of 7% MA or
17% EDTA followed by 0.2% CTR or 2% CHX showed
100% residual and antimicrobial activity. Conclusions:
A 0.2% CTR solution alone and the combinations in
which 2% CHX or 0.2% CTR was the final irrigating solu-
tion achieved the maximum residual and antimicrobial
activity. (J Endod 2011;37:363–366)
Key Words
Biofilms, cetrimide, chlorhexidine, EDTA, Entero-
coccus faecalis, maleic acid, sodium hypochlorite
From the Departments of *Preventive and Operative
Dentistry and †Microbiology, School of Dentistry, University of
Granada, Granada, Spain.
Supported by the Research Group CTS-167 of the Junta de
Andaluc
ıa, Spain.
Address requests for reprints to Dr Mar
ıa Teresa Arias-Moliz,
Department of Microbiology, School of Dentistry, Campus de
Cartuja, Colegio M
aximo s/n E-18071, Granada, Spain. E-mail
address: mtarias@ugr.es
0099-2399/$ - see front matter
Copyright ª 2011 American Association of Endodontists.
doi:10.1016/j.joen.2010.11.036
JOE — Volume 37, Number 3, March 2011
E nterococcus faecalis is a commonly isolated species from persistent apical perio-
dontitis (1–3). Some possible factors facilitating its long-term survival in the root
canal system are its ability to adhere to dentin and invade dentinal tubules (4, 5) and to
form communities organized in biofilms, which may contribute to bacterial resistance
and persistence after intracanal antimicrobial procedures (6).
Bacterial elimination from the root canal is achieved by means of the mechanical
action of instruments and irrigation as well as the antibacterial effects of the irrigating
solutions. Nowadays, most studies focus on the antimicrobial properties of the irrigating
solutions, involving both forms of bacterial growth, planktonic and biofilm (7).
However, few studies look into the residual antibacterial activity and its influence on
microbial adhesion to the dentin surface (8, 9). This is a relevant aspect because
microbial adherence to the dentin is the first step in colonization, including tubule
invasion, and the origin of biofilm infections.
Sodium hypochlorite (NaOCl) is a frequently used irrigating solution in endodon-
tics because of its ability to dissolve necrotic tissue as well as its potent antimicrobial
action (7, 10, 11). However, it has not been reported to have any residual
antimicrobial activity (12, 13). Other irrigating solutions such as chlorhexidine
(CHX) and cetrimide (CTR) are less effective than NaOCl in eradicating E. faecalis
biofilm (11, 14), but CHX has substantive properties and is able to inhibit dentin
adherence of certain bacteria (13).
Chelating agents are used to remove the smear layer produced during mechanical
instrumentation (7). Although EDTA is one of the most commonly used, its antimicrobial
activity against biofilms is a matter of some controversy (9, 11, 15). Maleic acid (MA),
a mild organic acid, has been more recently proposed for use as a final irrigating
solution, as an alternative to EDTA (16), because of its better smear layer removal
from the apical third of the root canal system (17) and its lower toxicity (16). Further-
more, its antibacterial activity has been shown in vitro against E. faecalis biofilm (18).
Different protocols and/or combinations of irrigating solutions are used in the
final irrigation of the root canals, but their residual activity is not well known. Therefore,
the objective of this study was to evaluate the residual antimicrobial activity and the
capacity to eradicate E. faecalis biofilm using several irrigating solutions, alone and
in combination, in a dentin-volumetric test.
Materials and Methods
Bacteria Strain and Irrigating Solutions
The bacteria strain used in this study was E. faecalis ATCC 29212, which was taken
from a 4 C stock culture and streaked out twice on brain-Heart Infusion (BHI) (Schar-
lau Chemie SA, Barcelona, Spain) agar plates for 24 hours at 37 C. From the subculture
of E. faecalis, a 1 McFarland standard suspension was prepared in BHI and then diluted
30-fold to obtain an initial bacterial suspension of approximately 1  107 colony-
forming units per milliliter (CFU/mL).
The final irrigating solutions tested were: 2.5% NaOCl (Panreac Qu
ımica SA, Cas-
tellar del Valles, Spain), 2% CHX (Guinama, Alboraya, Spain), 0.2% CTR (Sigma-Aldrich
Chemie, Steinheim, Germany), 17% EDTA (Merck, Darmstadt, Germany), 7% MA (Pan-
reac Qu
ımica SA, Castellar del Valles, Spain), combinations of 2.5% NaOCl followed by
17% EDTA or 7% MA, and combinations of 2.5% NaOCl followed first by 17% EDTA or
7% MA and then by 0.2% CTR or 2% CHX.
Final Irrigation Residual and Antimicrobial Activity 363
Basic Research—Technology
Preparation of Dentin Blocks
Eighty noncarious, unrestored freshly extracted human molars,
stored in 0.1% thymol solution at 4 C, were randomly divided into eight
groups (n = 10): four groups for residual activity (A-D) and four
groups for antimicrobial activity (E-H). The teeth were sectioned using
an Accuton-50 machine (Struers, Copenhagen, Denmark) at a high
force of 3,200 rpm without rotation and abundant water cooling. The
two apical thirds of the roots were discarded as well as the occlusal
coronal enamel in order to obtain a flat coronal dentin surface. This
slice was cut into serial blocks. Dentin blocks without enamel were
selected from each tooth and randomly assigned to the protocols listed
in Table 1. The block size was adjusted using a calibrator and polished
with 150-, 220-, and 600-grit silicon carbide papers in ascending order
to obtain 2  2  1.8 mm (width  length  height) specimens. The
smear layer formed during preparation of the dentin blocks was elim-
inated by submerging them in 17% EDTA for 2 minutes and then in 2.5%
NaOCl for 1 minute to standardize the specimens before their steriliza-
tion and separation into the different groups for study. After sterilization,
the specimens were incubated in BHI for 24 hours at 37 C to ensure no
bacterial contamination. The dentin blocks were then kept in sterile
saline solution until used.
Residual Antimicrobial Activity Test
Sterile dentin blocks were submerged in 100 mL of the different
irrigating solutions following the protocols in Table 1 in a 96-well
microtiter plate (Nunclon Delta Surface; Nunc, Roskilde, Denmark).
The dentin blocks were transferred to a microtiter plate with 180 mL/
well of the initial bacterial suspension and then incubated for 24
hours at 37 C on a rocking table (Swing Sw 8 10000-00015;
OVAN, Badalona, Spain) at five rocks per minute. One nontreated
dentin block per tooth was inoculated in 180 mL of the initial bacte-
rial suspension as the positive control, whereas 10 nontreated dentin
blocks inoculated in 180 mL of BHI served as the sterility controls.
After incubation, the specimens were rinsed once with 180 mL of
0.9% saline solution for 2 minutes. The specimens were then placed
in a microtiter recovery plate with 200 mL BHI per well and soni-
cated on a water-table sonicator (Model 5510E–MT; Branson, Dan-
bury, CT) for 10 minutes. Disrupted biofilm cultures were diluted
serially and plated for viable cell counting.
Biofilm Antimicrobial Activity Test
The wells of a 96-well microtiter plate were inoculated with 180 mL
of the initial bacterial suspension, whereas 10 wells were inoculated
with sterile BHI for the sterility control. The sterile dentin blocks
were submerged in the inoculated wells, and they were incubated on
the rocking table for 3 weeks at 37 C with 95% relative humidity.
The BHI was refreshed daily to ensure the growth of E. faecalis on
the dentin blocks, and the purity of the cultures was checked at regular
intervals. Dentin specimens with the biofilms were rinsed with 180 mL
0.9% saline solution for 2 minutes.
The antimicrobial activity assay was performed in a microtiter
plate with 100 mL of the irrigating solutions per well. The first and
the last wells of each row, containing BHI, served as the sterility (dentin
blocks without bacteria) and growth controls, respectively. The saline-
rinsed specimens were then added to the wells. After subjecting the
dentin blocks to the irrigation protocols, sterile absorbent paper disks
(IVD; Becton, Dickinson and Company, Sparks, MD) were used to elim-
inate any excess solution from the specimens. Afterwards, they were
placed in Eppendorf tubes with 200 mL BHI, vortexed for 2 seconds,
and then sonicated for 10 minutes to assure biofilm recovery. Viable
cell counting was performed as previously described.
Results of E. faecalis biofilm residual activity and antimicrobial
activity by different protocols were, respectively, expressed as the inhi-
bition percentage of biofilm formation and the kill percentage of biofilm
and calculated for each group of teeth (A-H) as follows: (1 – [mean
CFUprotocol/mean CFUcontrol])  100). The term ‘‘eradication’’ was
used to denote the kill of 100% of the bacterial population. To compare
the efficacies of the different protocols when the kill percentage varied
from 100%, as well as the reduction percentage related to the control,
the Student t test was used, previously subjecting data to the Anscombe
transformation (19).
Results
All negative controls showed no bacterial growth. The results of the
residual antimicrobial activity and antimicrobial activity tests are shown
in Table 1 and Figure 1. The 2% CHX and 0.2% CTR applied alone for 1
minute or as the final irrigation of a previous sequence of 2.5% NaOCl/
17% EDTA and 2.5% NaOCl/7% MA completely inhibited the 24-hour
E. faecalis biofilm formation. With respect to the control, 2.5% NaOCl

TABLE 1. Inhibition of E. faecalis Biofilm Formation (residual activity) and Antimicrobial Activity against E. faecalis Biofilm of Different Irrigating Solutions
Inhibition of E. faecalis biofilm formation Antimicrobial activity against
(residual activity) E. faecalis biofilm

Inhibition Kill percentage

Irrigation Units without percentage mean Units without mean ± standard
protocol biofilm formation ± standard deviation biofilm eradication deviation*
2.5% NaOCl 0/10 18.10  41.49a1 0/10 100
2% CHX 10/10 100 5/10 99.93  0.09a2
0.2% CTR 10/10 100 0/10 100
17% EDTA 0/10 64.20  28.09b2 10/10 44.00  14.11b2
7% MA 0/10 85.86  11.56c2 3/10 99.99  0.00a2
2.5% NaOCl/17% EDTA 0/10 21.31  54.12a1 0/10 100
2.5% NaOCl/17% EDTA/2% CHX 10/10 100 0/10 100
2.5% NaOCl/17% EDTA/0.2% CTR 10/10 100 0/10 100
2.5% NaOCl/7% MA 0/10 34.17  48.14a1 0/10 100
2.5% NaOCl/7% MA/2% CHX 10/10 100 0/10 100
2.5% NaOCl/7% MA/0.2% CTR 10/10 100 0/10 100
NaOCl, sodium hypochlorite; CHX, chlorhexidine; CTR, cetrimide; EDTA, ethylenediaminetetraacetic; MA, maleic acid.
Read vertically, the same letters show differences not statistically significant.
Read vertically, number 1 means inhibition percentage or kill percentage with nonsignificant differences calculated with respect to the control. Number 2 means statistically significant differences.
*Biofilm reduction percentage calculated only in units without biofilm eradication.

364 Baca et al. JOE — Volume 37, Number 3, March 2011


Figure 1. SEM images of the inhibition of E. faecalis biofilm formation (residual activity) with final irrigation regimens on dentin blocks. (A) 2.5% NaOCl, (B)
17% EDTA, (C) 7% MA, and (D) 2.5% NaOCl/7% MA/0.2% CTR.
reduced biofilm formation (18.10%) but without statistical signifi-
cance, 17% EDTA reduced biofilm significantly by 64.21% (p <
.001), and 7% MA was 85.86% effective (p < .001). The latter two,
when applied after NaOCl, exerted a much lesser effect.
The 2.5% NaOCl applied for 1 minute alone or in any of the combi-
nations tested effectively eradicated the 3-week E. faecalis biofilm. This
same result was observed with 0.2% CTR. The biofilm was eradicated by
7% MA and 2% CHX in 7 of 10 and 5 of 10 dentin blocks, respectively,
and the percentage of kill in the remainder of the samples was over
99%. Although 17% EDTA was unable to eradicate biofilm in any dentin
blocks, it achieved a kill percentage of 44%.
Discussion
Our objective was to assay different irrigating solutions, alone or in
combination, to determine their ability to inhibit biofilm formation as
well as their antimicrobial activity against a 3-week biofilm. Both prop-
erties are desirable in order to control dentin infection and delay rein-
fection of the root canal.
Dentin was selected because it represents the primary substratum
for bacterial adhesion and biofilm formation (20), and its interaction
with endodontic irrigating solutions has been clearly shown (21).
Based on previous models, we designed a new dentin-volumetric test
as a carrier or biological unit of biofilm formation. The specimens
are easily size standardized and infected, and they are simple to manip-
ulate. At least four specimens can be obtained from each molar, which
permits their assignment to different groups, reducing the inherent vari-
ability of the sample. The irrigating solutions used, three antimicrobial
agents (NaOCl, CHX, and CTR) and two chelating agents (EDTA and
MA), were tested alone and in different combinations that could be
proposed as a final irrigation regimen. One minute of time is considered
adequate for such regimens (22).
JOE — Volume 37, Number 3, March 2011
Basic Research—Technology
Although the mechanism of bacterial invasion is not completely
understood, bacterial adhesion to dentin is the first step in the process
(5). Here, residual antimicrobial activity was evaluated as the ability to
inhibit 24-hour biofilm formation because adherence has been shown
to occur between bacteria and substrate in this time period (8). The
results of this study show that CTR and CHX alone or as the final agent
within the protocol of irrigation can fully inhibit biofilm formation. In
contrast, NaOCl exerted no residual action, and MA and EDTA alone
or after the application of NaOCl presented intermediate values. Such
findings could be explained by the mechanism of action of the solutions,
and in particular their antimicrobial activity, wettability, and/or demon-
strated ability to alter the physicochemical properties of dentin (8).
Our results agree with previous studies reporting that NaOCl has
little residual antimicrobial activity (12). Its antimicrobial effect depends
on the free chlorine available in the solution and its high surface tension
(23), which would impede its penetration into the dentin. Moreover,
NaOCl may be inactivated by the buffer ability of dentin (21).
The high and prolonged substantivity of CHX, which allows it to be
slowly released in an active form (24), may have fully inhibited the
formation of E. faecalis biofilm on dentin. This finding agrees with
a previous ex vivo study in which CHX diminished adherence of E. fae-
calis to dentin (8). CTR is a cationic surfactant with proven bactericidal
activity against E. faecalis biofilm (14). Its substantivity in dentin has
not been demonstrated to date; yet when combined with CHX in Cetrex-
idin (0.2% CHX + 0.2% CTR; GABA Vebas, San Giuliano Milanese, Italy)
the mixture displayed residual antibacterial activity (25). Our results
show that CTR exerts a residual antibacterial activity comparable to
CHX. This could stem from its cationic nature, which lends it the
capacity to interact with the dentin, resulting in an action similar to
that of CHX.
The chelating agents tested, 7% MA and 17% EDTA, attained
a significant percentage of inhibition of E. faecalis biofilm formation
Final Irrigation Residual and Antimicrobial Activity 365
Basic Research—Technology
(85% and 64%, respectively). Both of them cause demineralization of
dentin, which exposes the collagen (26, 27) and contributes to create
an ideal substrate for E. faecalis adherence (28). Furthermore, the
greater residual activity of MA as compared with EDTA can be attributed
to its stronger antimicrobial action (11, 18).
The results of the antimicrobial activity assay showed that NaOCl
and CTR were the most effective agents in eradicating 3-week E. faecalis
biofilms after 1-minute contact time, and their action proved signifi-
cantly better than CHX, a finding concordant with previous studies
(11, 29). Because the MA antimicrobial activity had not been tested
previously on dentin, we underline that MA eradicated the biofilm in
7 of 10 specimens; the remaining three specimens showed a kill
percentage of 99.99%. These findings are consistent with its reported
capacity to eradicate E. faecalis biofilm in vitro (18). Such activity
might reside in the chemical nature of MA; as an organic acid (pH =
1.28), it decreases the internal pH of the microbial cell and alters
cell membrane permeability (30). EDTA achieved 44% kill in our study,
possibly because of its calcium-chelating effect, which facilitates
mechanical removal of bacteria and can also affect metabolic pathways
in the bacterial cell (31). All the combinations tested eradicated E. fae-
calis biofilms from the dentin blocks, presumably because of the anti-
microbial action of NaOCl and its additional action with CHX or CTR in
the different protocols.
The dentin volumetric test developed in this study has shown itself
to be an adequate method for assessing the antimicrobial properties of
root canal irrigating. From the clinical standpoint, the high degree of
residual action seen with CHX and CTR suggests their use as final irri-
gating agents. CTR was the only irrigating solution that eradicated bio-
film and achieved maximal residual activity.
Acknowledgments
The authors thank Francisca Castillo P
erez for her technical
assistance.
The authors deny any conflicts of interest related to this study.
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