Basic Research—Technology
Validation of Biofilm Assays to Assess Antibiofilm
Efficacy in Instrumented Root Canals after Syringe
Irrigation and Sonic Agitation
Anil Kishen, BDS, MDS, PhD,* Annie Shrestha, BDS, MSc, PhD,*
and Aldo Del Carpio-Perochena, DDS, PhD†
Abstract
Introduction: Different methods to characterize bacte-
rial biofilms have been established, each presenting
with distinct advantages and shortcomings. The aim of
this study was to validate the ability of microbiological
culture, the adenosine-50 -triphosphate (luminescence)
assay, and molecular and microscopic methods to assess
antibiofilm efficacy. Methods: Thirty-nine extracted
single-rooted teeth were selected. Enterococcus fae-
calis biofilms were grown for 21 days and randomly
distributed into 3 groups. All canals were instrumented
(F3 ProTaper Universal; Dentsply Sirona, Johnson City,
TN) and irrigated (ProRinse needles, Dentsply Sirona)
as follows: group 1, sodium hypochlorite and EDTA irri-
gation; group 2, supplemented with sonic agitation of
NaOCl, and group 3, sterile distilled water irrigation.
Bacteriologic samples were collected before (S1) and af-
ter canal preparation (S2) and subjected to quantifica-
tion by culture methods, quantitative reverse
transcriptase real-time PCR (qRT-PCR), and lumines-
cence assay. The biofilm structure and bacterial cell
viability were evaluated under scanning electron micro-
scopy (SEM) and confocal laser scanning microscopy
(CLSM). Data were subjected to statistical analysis to
determine the statistical significance (P < .05). Results:
S1 samples showed approximately 8-log colony-
forming-units of bacteria using both culture and qRT-
PCR. The reduction in bacterial populations and relative
luminescence was highly significant in the S2 samples
from groups 1 and 2 (P < .001). SEM and CLSM showed
well-matured root canal biofilms in the pretreatment
samples that were reduced after treatment. Irrigation
with NaOCl combined with sonic agitation significantly
decreased the percentage of live cells (P < .05) but
was not able to eliminate the biofilm structure. Conclu-
sions: This study highlighted the maximum reduction of
microbes after instrumentation-syringe irrigation.
Although supplementary sonic agitation reduced the
From the *Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada; †Department of Endodontics, Faculty of Dentistry, University of Manitoba, Winnipeg,
Manitoba, Canada.
Address requests for reprints to Dr Anil Kishen, Discipline of Endodontics, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON, M5G 1G6,
Canada. E-mail address: anil.kishen@utoronto.ca
0099-2399/$ - see front matter
Copyright ª 2017 American Association of Endodontists.
https://doi.org/10.1016/j.joen.2017.10.005
JOE — Volume -, Number -, - 2017
root canal biofilm further, it did not completely eliminate the biofilm from a single
root canal model. The merits of combining microbiological and molecular quantification
methods with CLSM for the comprehensive assessment of antibiofilm efficacy in root
canals were emphasized. (J Endod 2017;-:1–7)
Key Words
Biofilm, culture, detection techniques, microscopy, polymerase chain reaction
A biofilm is a complex
biological community
consisting of bacterial col-
Significance
This study highlighted the maximum reduction of
microbes after instrumentation-syringe irrigation.
onies and extracellular
Supplementary sonic agitation reduced the biofilm
polysaccharides (EPSs),
contents further but did not completely eliminate
giving it the characteristics
the biofilm in the tested single-canal models. The
of a multicellular system.
benefit of combining microbiological and molecu-
Microbiological assays that
lar quantification methods with microscopy for
effectually characterize bio-
the comprehensive assessment of antibiofilm effi-
film bacteria have been a
cacy in root canals was emphasized.
challenging subject in end-
odontic microbiology (1);
yet, this is a cardinal step used routinely for the estimation of microbial biomass
while assessing the efficacy of different antimicrobial irrigants/irrigation strategies
in endodontics. Current biofilm assays characterize features such as the number/
type of resident microbes, vitality (dead/living cells) of resident microbes, thickness,
structure (homogeneous, irregular, dense, or porous), topography, and biomass or
EPS levels (1, 2). Apparently, every assay provides distinctly different information,
and, therefore, depending on a sole method for biofilm characterization would be
inadequate.
Biofilm evaluation is an essential step for assessing antibacterial efficacy in root
canal disinfection (2–4). Direct enumerations of colony-forming units (CFUs) using
culture-based methods have been a gold standard in bacterial detection/quantification.
However, culture methods have been suspected because of their inability to detect viable
but nonculturable bacteria (4, 5) and the need for a prolonged incubation time with
multistep culture techniques (2). This approach is further marred by the limitations
of sampling from root canals (6). The specificity of culture medium, transport medium,
toxicity of by-products, and metabolic dependency in multispecies biofilm could
further amplify the limitations of cultivation methods (5). Accordingly, microscopic,
Antibiofilm Efficacy in Root Canals 1
Basic Research—Technology
biochemical, and molecular techniques have been advocated to reliably
estimate bacterial biofilms.
Polymerase chain reaction (PCR) is a highly sensitive molecular
method used for microbial detection in root canals (5, 7). A real-
time PCR (also known as quantitative PCR [qPCR]) is used to amplify
and simultaneously quantify a targeted DNA molecule, enabling both
detection and quantification of the specific DNA sequences (4, 8).
Real-time PCR is a distinct technique that uses reverse transcription
to quantify messenger/noncoding RNA. Quantitative reverse transcrip-
tase real-time PCR (qRT-PCR) effectively quantified the number of
RNA transcripts of specific genes from bacteria growing in biofilms.
qRT-PCR is a sensitive method to quantify gene expression from biofilms
in which only a small amount of the sample is available (8). qPCR is
widely used in endodontics to assess the degree of disinfection achieved
after root cleaning with topical antimicrobials as well as shaping (9–
11).
Scanning electron microscopy (SEM) and transmission electron
microscopy have been effective workhorses for the characterization
of the biofilm structure (2). The main disadvantage with these tech-
niques is the prerequisite for extensive sample preparations, which
could affect the original biofilm morphology. Recently, confocal laser
scanning microscopy (CLSM) in combination with specific fluores-
cence stains that monitor growth and metabolic activities of bacteria
has become a versatile tool for biofilm examination (12, 13).
However, microscopy offers limited specificity and sensitivity to detect
microbes from clinical samples (14). The current study aimed to
compare the capabilities of conventional microbiological quantification
techniques with microscopic techniques to assess the antibiofilm effi-
cacy after instrumentation-syringe irrigation and supplementary sonic
activation of sodium hypochlorite (NaOCl) from an in vitro root canal
model. This investigation offers information on the degree of microbial/
biofilm reduction achieved by root canal instrumentation–syringe irri-
gation (stage 1) and after sonic agitation of NaOCl (stage 2) and the
appropriateness of different biofilm assays to assess the antibiofilm
efficacy in root canals.
Materials and Methods
Specimen Preparation
Freshly extracted single-rooted teeth without caries and fractures
were included in the study after University of Toronto Ethics Board
approval. Thirty-nine teeth were selected and autoclaved for steriliza-
tion. The teeth were decoronated and standardized to 12-mm-length
specimens. Root canals in all the teeth were enlarged with ISO size
20 K-files up to an 11-mm working length (WL). The root canals
were irrigated with 6 mL 5.25% NaOCl, 2 mL 17% EDTA, and 2 mL
5.25% NaOCl. NaOCl was neutralized with filter-sterilized 10% sodium
thiosulfate. Each of the prepared roots was immersed in 1 mL brain-
heart infusion (BHI) broth and autoclaved for sterilization. All the
chemicals used in this study were of analytical grade and were pur-
chased from Sigma-Aldrich (St Louis, MO) unless noted otherwise.
Root Canal Biofilm Formation
Overnight cultures of Enterococcus faecalis (ATCC 29212; Amer-
ican Type Culture Collection, Manassas, VA) were grown in BHI broth at
37 C and adjusted to 108 CFU/mL (optical density at 600 nm = 1). The
prepared root canals were kept in sterile centrifuge tubes, and 0.5 mL
bacterial culture was added. One hundred microliters of the culture was
added inside the root canal lumen, and the tubes were centrifuged at
1400g, 2000g, 3600g, and 5600g in a sequence for 5 minutes. A fresh
solution of bacteria was added between every centrifugation. All tubes
with root specimens were then incubated at 37 C for 21 days, changing
2 Kishen et al.
the media every 72 hours. Teeth were randomly divided into 2 experi-
mental groups of 12 teeth each according to the irrigation technique
used and a control group consisting of 15 teeth. Of the 15 teeth from
the control group, 3 samples were processed to evaluate the initial bio-
film biomass using confocal microscopy.
The root specimens were removed from the tubes, and the external
surfaces were wiped with sterile gauge. The apical foramen was sealed
with epoxy resin. An initial microbial sample of the root canal (S1) was
taken. Canals were filled with 1 mL sterile phosphate-buffered saline
(PBS) solution and gently filed using ISO size 20 K-files. The S1 sample
was obtained by the sequential use of 3 paper points placed to the WL.
Each paper point remained in the canal for 30 seconds before they were
transferred to the tubes containing 4 mL BHI broth for further analysis.
Treatment Groups
The root canals were sequentially enlarged using ProTaper Univer-
sal instruments (Dentsply Sirona, Johnson City, TN) at 300 rpm and
200 g/cm torque (ProMark Endodontic Motor, Dentsply Sirona) up
to F3 at the WL. The samples were irrigated with 1 mL NaOCl using a
5-mL syringe and 30-G ProRinse endodontic irrigation probes (Dents-
ply Sirona) placed in the canal 2 mm short of the WL between successive
instruments. After instrumentation, NaOCl was left undisturbed in the
canal for 60 seconds, and the final irrigation procedure was performed
as follows:
1. Group 1 (syringe irrigation group): 2.5 mL NaOCl, 5 mL EDTA, and
2.5 mL NaOCl.
2. Group 2 (Sonic agitation group): sonic agitation using the EndoAc-
tivator (Dentsply Sirona) was applied on the canals filled with EDTA
(60 seconds) and NaOCl (30 seconds); a #25/0.04 noncutting poly-
mer tip of the EndoActivator placed 2 mm from the WL was used for
each irrigation cycle (15). Overall, 20 mL irrigant was used per ca-
nal in both groups for approximately the same time period. After the
final irrigation, the root canals were irrigated with 2 mL sodium thio-
sulfate to inactivate residual NaOCl followed by 2 mL distilled water
and dried with sterile paper points.
3. Group 3 (control group): root canals were shaped in the same
manner as in group 1 with sterile distilled water used as an irrigant
(a total of 20 mL per canal).
The duration for irrigation in all groups was standardized to be
approximately 10 minutes. Six specimens from each group were used
for microbiological quantification, and the remaining 6 specimens
were used for CLSM and SEM.
Sampling and Processing
Bacteriologic samples collected after canal preparation (S2) were
taken using an F4 file (n = 6/group). Canals filled with PBS were
enlarged using an F4 file as a hand instrument. The file with dentin shav-
ings was transferred to tubes containing 4 mL BHI broth. A medium-
sized paper point was agitated inside the canals, left for 30 seconds
to soak up the canal contents, and transferred into the tube along
with the file. The BHI broth with the paper points and files was incubated
for 4 hours at 37 C to provide an enrichment phase for the bacteria
(16). This time duration was chosen based on the bacterial growth
in the S2 samples of group 3 to attain an optical density of 0.1.
Quantification by Culture and PCR
The samples were agitated in a vortex mixer for 1 minute and
serially diluted (10-fold) in PBS. Aliquots of 1 mL were plated onto
BHI plates and incubated at 37 C for 48 hours. The CFUs grown
JOE — Volume -, Number -, - 2017

were counted and later transformed into log CFU values based on the
known dilution factors.
qPCR was used to quantify the following previously published pro-
tocol (17). In brief, genomic DNA (gDNA) was extracted from 2 mL of
the sample volume using the QIAamp DNA Mini Kit (Qiagen, Valencia,
CA) according to the manufacturer’s instructions. To maximize gDNA
extraction, a step of preincubation with lysozyme for 30 minutes was
added. gDNA extracts were frozen at 20 C until qPCR analysis. Sam-
ples were brought to room temperature, and DNA was extracted using
the QIAamp DNA Mini Kit following the protocol recommended by the
manufacturer.
To quantify E. faecalis cells in root canal samples, 16S ribosomal
RNA gene-targeted qPCR was performed with Power SYBR Green PCR
Master Mix (Applied Biosystems, Foster City, CA) on an ABI 7500
real-time PCR instrument (Applied Biosystems) in a total reaction vol-
ume of 20 mL. Species-specific primers for E. faecalis were used ac-
cording to a previous study (17). Primer pairs (50 -30 ) (GTT TAT GCC
GCA TGG CAT AAG AG and CCG TCA GGG GAC GTT CAG) in a concentra-
tion of 0.5 mmol/L each and a DNA extract volume of 2 mL were added to
the PCR Master Mix in MicroAmp Optical 96-well reaction plates
(Thermo Fisher, Mississauga, ON, Canada). The cycling conditions
for qPCR included 95 C for 10 minutes and 40 repeats of the following
steps: 95 C for 1 minute, 60 C for 1 minute, and 72 C for 1 minute. The
double-stranded DNA product was measured at 78 C. At each cycle, the
accumulation of PCR products was detected by monitoring the increase
in fluorescence of the reporter dye (double-stranded DNA-binding
SYBR Green). All measurements were performed in duplicate for sam-
ples and triplicate for standards. In all experiments, triplicates of the
appropriate negative controls containing no template DNA were sub-
jected to the same procedures to exclude or detect any possible contam-
ination or carryover. After amplification, melting curve analysis of the
PCR products was performed to determine the specificity of the ampli-
fied products. The melting curve was obtained from 60 C–95 C, with
continuous fluorescence measurements taken at every 1% increase in
temperature. Data acquisition and analysis were performed using Abi
7500 software v2.0.4 (Applied Biosystems). E. faecalis cell counts
were calculated for each sample based on the obtained standard curves
(10-log–fold standard curve) for direct bacterial quantification. To
obtain the standard curve, gDNA was isolated from a fresh pure culture
of E. faecalis and diluted serially, analyzed using qPCR, and quantified
using the plating method.
Adenosine-50 -triphosphate Assay
The bacterial activity was measured quantitatively using an adeno-
sine-50 -triphosphate (ATP) assay (Bactiter Glo reagent; Promega, Mad-
ison, WI) following the manufacturer’s protocol. In brief, 100 mL
bacterial suspension obtained from the root canal was added in an opa-
que white 96-well plate (Greiner, Monroe, NC). The Bactiter Glo reagent
(100 mL) was added in each well and incubated for 5 minutes, and
luminescence was measured using a luminometer (Synergy H1 Multi-
Mode Reader; Biotek, Winooski, VT).
SEM
The roots were divided longitudinally with a scalpel positioned on
the grooves prepared previously and fixed overnight in 2.5% glutaral-
dehyde in 0.1 mol/L phosphate buffer at 4 C (n = 3). The specimens
were serially dehydrated using ethanol; critical point dried in a critical
point drier (CPD030; BalTec, Balzers, Liechtenstein); sputter coated
with palladium (SCD005, BalTec); and examined using SEM at magni-
fications of 1000, 3000, and 5000. Each sample was imaged at 9
random locations along the root canal lumen.
JOE — Volume -, Number -, - 2017
Basic Research—Technology
CLSM
The roots were divided longitudinally with a scalpel positioned on
the grooves prepared on either side of the root (n = 3). The root halves
were stained with Live/Dead BacLight Bacterial Viability (Invitrogen, Eu-
gene, OR) (excitation/emission 495/515 nm) and imaged using a
confocal laser scanning microscope (TCS-SPE; Leica Microsystems,
Mannheim, Germany). Three ‘‘stacks’’ of the apical and medium thirds
(96 images in total) were scanned at 20 oil lens zoom 2, 1-mm step
size, and a format of 512  512 pixels. The area of each image repre-
sented 275  275 mm. Bioimage_L (Luis Ch
avez de Paz, Farmington,
CT) was used to calculate the biovolume (mm3) and bacterial viability
(%) of the cells from the confocal laser scanning microscopic images
(18).
Statistical Analysis
The average of the bacterial counts was calculated. Data concerning
bacterial viability were evaluated for statistical significance using the
1-way analysis of variance test followed by the Tukey test for multiple
comparisons. The nonparametric Kruskal-Wallis and Dunn tests
(P < .05) were used to perform multiple comparisons of the biofilm vol-
umes obtained from CLSM because the data did not show a normal dis-
tribution. The significance level was fixed at P < .05. Prisma 6.0
(GraphPad Software Inc, La Jolla, CA) was used as the analytical software.
Results
Quantification by Culture and PCR
The S1 samples showed approximately 8-log CFU biofilm bacteria
using both culture and qPCR methods. After instrumentation and 2
different final irrigation methods, the S2 samples showed more than
a 4-log reduction of the initial bacterial load (P < .05). Irrigation
with/without sonic agitation did not display any significant difference
between the culture and qPCR methods (P > .05). Group 3 showed
no significant bacterial reduction between the S1 and S2 samples.
Figure 1. A comparison of bacterial quantification from root canals at various
stages of cleaning and shaping based on microbiological culture and molec-
ular methods. Samples obtained before irrigation and instrumentation (S1)
and after irrigation and instrumentation (S2). Quantification using microbio-
logical culture correlated with molecular quantification using qPCR. The S1
samples from all 3 groups and 2 detection methods showed similar bacterial
counts. Disinfection with and without sonic agitation resulted in a significant
reduction of viable bacteria in S2 samples compared with S1 samples
(*P < .05). In group 3 wherein disinfection was performed using sterile saline,
a minimal reduction of viable bacteria was noted. The error bars show the
standard deviation from the average value.
Antibiofilm Efficacy in Root Canals 3
Basic Research—Technology
Figure 1 shows the quantified bacteria obtained from microbiological
culture and qPCR.
ATP Assay
Bacteria from S1 samples in all 3 groups showed high ATP read-
ings. In groups 1 and 2, luminescence signals in the S2 samples were
significantly low (P < .001) compared with the S2 samples in group
3. The ATP readings in the S2 samples were relatively lower when
compared with the quantification based on the culture and qPCR
methods. Table 1 shows the relative luminescence units obtained
from the S1 and S2 samples in all 3 groups.
SEM
Uniformly thick matlike E. faecalis biofilms with an abundant
polymeric matrix were found adherent on the root canal dentin surface
(Fig. 2A–C). Shaping and irrigation with the syringe method resulted in
an uneven disruption of the biofilm (Fig. 2D–F). Areas of clean and
open dentinal tubules as well as areas with a residual biofilm were
observed. Sonic agitation in addition to instrumentation-syringe irriga-
tion resulted in a cleaner dentin surface but with regions of a residual
biofilm on the root canal walls (Fig. 2G–I).
CLSM
Representative images from each sample were taken with a total of
8 microphotographs (4 of the middle third and 4 of the apical third).
Uniformly thick biofilms composed of both live and dead cells were
found along the root canal walls (Fig. 3A). The biofilms retained the
structure and composition in group 3 (distilled water) (Fig. 3B),
whereas treatment groups 1 and 2 showed disrupted biofilm structures
with mostly dead bacterial cells (Fig. 3C and D). Data were pooled to
provide a single median because of the nonparametric distribution of
the values. The conventional disinfection protocols were efficient to re-
move surface-adherent biofilms regardless of the use of sonic agitation
with a reduction in the overall biofilm height (P > .05) (Fig. 3E and F).
The bacterial volume and viability of group 3, which was irrigated with
distilled water, did not show statistical differences compared with the no
treatment group (control) (P > .05) (Fig. 3G and H). Supplementary
irrigation with NaOCl combined with sonic agitation significantly
decreased the percentage of live cells in the biofilm structure adherent
on the root canal dentin (P < .05).
Discussion
During the biofilm assessment, the determination of microbial
CFUs quantified the cultivable bacteria only, whereas molecular tech-
niques based on nucleic acid measurements are generally more sensi-
tive than CFU-based methods (5, 19–21). Molecular methods such as
PCR could detect residual DNA and messenger RNA up to 30 hours after
bacterial death as confirmed by culture methods (20, 21). This point is
particularly significant when antibacterial agents are being evaluated.
TABLE 1. Bacterial Adenosine-50 -triphosphate Activity Quantified Using the Luminescence Assay
Group 1
S1 S2 S1
63,735 655.66*,† 45,296.67
(8062.12) (148.92) (18,447.83)
ATP measurements were calculated as the average of triplicate readings, and the numbers in parentheses are the standard deviation. Luminescence was calculated as the mean of the signals from the bacterial
samples as expressed in relative light units (RLUs).
*A statistically significant reduction in RLUs was noted from S1 to S2 in each group (P < .001).
†
S2 samples from groups 1 and 2 showed a significant difference from group 3 (P < .05).
4 Kishen et al.
Thus, a clear-cut consensus is required on the appropriateness of
different biofilm assessment techniques to determine the effectiveness
of topical antimicrobials in root canals.
Phenotypic characteristics of resident bacteria, the ultrastructure
of the biofilm, and the constituents of the biofilm matrix are factors that
contribute to challenges in biofilm assessment (2). To circumvent these
challenges, several protocols recommended the removal of biofilm bac-
teria from the substrate by sonication or centrifugation before using the
supernatant for any quantitative analysis (2). While assessing biofilms
from the root canal, the recovery of post-treatment biofilm contents re-
mains to be a crucial step in this analysis. Microbes are sensitive to
different disinfectants, procedures, and shifts in growth conditions,
all of which would cause the bacteria to enter a slow growth (lag) phase.
In such situations, as in the current study, the biofilm bacteria have to be
grown in a nutrient-rich environment for a stipulated period of time
before they can be cultured and enumerated (2, 16, 22).
qPCR provides rapid, high-throughput detection and quantifica-
tion of target DNA sequences (19). It is also a better technique in terms
of avoiding cross contaminations because no additional manipulation
of samples is required after amplification. However, the phenotypic
and biochemical features must be confirmed from bacterial isolates us-
ing conventional culture techniques (23). qPCR has been adapted for
bacterial viability detection, which showed that RNA has low stability
because of its rapid degradation in dead cells. In addition, difficulties
associated with RNA isolation from samples would also contribute to
false-negative results, especially when low numbers of target cells are
expected. Considering these shortcomings, the correlation of cell
viability with persisting bacterial nucleic acid from infected tissue
must be well characterized before an appropriate amplification-based
analytical method can be adopted as a surrogate for more traditional
culture techniques (20).
In the current study, both the culture and PCR methods showed
approximately 8-log CFUs of biofilm bacteria, which reduced to almost
50% when instrumentation-syringe irrigation with NaOCl was used. Sup-
plementary disinfection with sonic agitation of NaOCl did not yield an
additional bacterial reduction. The ATP method is a sensitive bacterial
detection technique, yet, as in the culture and PCR techniques, it showed
a significant reduction in microbial loads subsequent to
instrumentation-syringe irrigation, with no statistically significant differ-
ence in the microbial reduction after sonic activation. Keeping in mind
the sampling techniques used in this study, it would be reasonable to
conclude that instrumentation-syringe irrigation produced the
maximum (and significant) reduction of microbes from the root canal
lumen.
The microscopic methods used in this investigation mainly as-
sessed the degree of reduction in the surface-adherent root canal bio-
film structures (2). SEM displayed that the combination of
instrumentation-syringe irrigation resulted in irregular areas of open
dentinal tubules and biofilm elimination. Complementing the
instrumentation-syringe irrigation with sonic agitation resulted in
Group 2 Group 3
S2 S1 S2
324.16*,† 61,605 12,764.66*,†
(70.59) (15,572.8) (1326.55)
JOE — Volume -, Number -, - 2017
 Basic Research—Technology

Figure 2. Surface topography of the bacterial biofilm on the root dentin surface was evaluated using SEM. (A–C) Scanning electron microscopic images of 3-week-
old E. faecalis biofilms on the root canal dentin surface. The biofilms presented as a uniformly thick matlike structure covering the entire dentin surface. The
biofilm showed an abundant polymeric matrix (area shown by the open arrow). (D–F) Conventional cleaning and shaping and syringe irrigation resulted in
the disruption of the biofilm. Areas of (E) clean and open dentinal tubules as well as with the (F) remaining biofilm were observed (closed arrow). (G–I) Sonic
agitation in addition to conventional disinfection resulted in a cleaner dentin surface. Aggregates of disrupted bacteria and EPSs were found on the dentin surface.

cleaner root canal dentin more consistently. Nonetheless, aggregates of
bacterial colonies and residual biofilm matrix were observed even after
sonic agitation of NaOCl.
CLSM is a nondestructive technique that does not require substantial
sample preparation, whereas SEM requires sample preparation steps that
would damage the biofilm matrix, leaving only the strongly adherent bac-
terial colonies on the dentin surface (24). The optical sections obtained
from CLSM reconstructed a 3-dimensional image of the entire biofilm,
whereas the application of nucleic acid–binding fluorescent dyes to estab-
lish cell status has become a routine practice for biofilm analysis (25).
Nevertheless, it was recently suggested that bacterial viability is strictly
the capacity to grow, and the fluorescent stain provides information on
bacterial vitality and not viability (25). In our study, CLSM revealed a
higher bacterial biomass and viable bacteria in controls, which decreased

JOE — Volume -, Number -, - 2017 Antibiofilm Efficacy in Root Canals 5

Basic Research—Technology

Figure 3. Confocal laser scanning microscopic representative images of the root canal lumen before and after different irrigation protocols. Bacterial biomass and viable cells
were higher in the (A) no treatment group and (B) group 3 (distilled water). Group 1 (syringe disinfection) and group 2 (with added sonic agitation) showed (C) markedly
decreased biofilm volume and (D) viable bacteria. The biofilm thickness of the no treatment control group and group 3 were in (E) a range of 56–90 mm, which significantly
decreased after the NaOCl disinfection protocols to (F) 10–30 um (P < .05). The medians and 25%–75% percentiles (in brackets) of (G) the bacterial volume and (H) the
percentage of live cells of biofilms adhered on the root canal dentin after different irrigation protocols. The different lowercase letters in each column represent significant
differences. CI, confidence interval; M, mean; Mdn, median; SD, standard deviation. (P < .05).

6 Kishen et al. JOE — Volume -, Number -, - 2017


markedly in the instrumentation-syringe irrigation group. Additional sonic
agitation of NaOCl resulted in an additional reduction in the residual vol-
ume and number of live bacteria from the root canal walls.
In a previous investigation, an in vitro multispecies anaerobic bio-
film grown under nutrient deprivation showed bacterial transformation
into a viable but nonculturable state and, under a nutrient-rich condi-
tion, returned to a culturable physiological state while it was still in a
biofilm (22). This indicated that CLSM combined with viability staining
reflected the ‘‘true viability status’’ of biofilm bacteria during starvation
rather than a culture-based method. This finding has to be taken into
account when considering results from solo culture-based investiga-
tions. Accordingly, it could be emphasized that microbiological
quantification methods should be complemented with fluorescent
image–based microscopy for an accurate assessment of biofilm (25).
In summary, this study emphasized the benefits of combining microbial
quantification assays with confocal microscopy–based biofilm
structural analysis for a comprehensive assessment of antibiofilm effi-
cacies in root canals. The findings also highlighted the ability of
instrumentation-syringe irrigation to achieve the maximum bacterial
reduction quantitatively from the root canal lumen, whereas additional
irrigation using sonic agitation of NaOCl further reduced surface-
adherent biofilm contents from the root canal wall.
Acknowledgments
Supported by a grant from the University of Toronto (A.K.)
(grant no. 928133).
The authors thank Ms. Martha Cordova for technical support.
The authors deny any conflicts of interest related to this study.
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