JOURNAL OF ENDODONTICS
Copyright 2002 by The American Association of Endodontists
The Effectiveness of Increased Apical Enlargement
in Reducing Intracanal Bacteria
Steven J. Card, DDS, MS, Asgeir Sigurdsson, DDS, MS, Dag rstavik, DDS, PhD, and
Martin Trope, DMD
It has been suggested that the apical portion of a
root canal is not adequately disinfected by typical
instrumentation regimens. The purpose of this
study was to determine whether instrumentation to
sizes larger than typically used would more effec-
tively remove culturable bacteria from the canal.
Forty patients with clinical and radiographic ev-
idence of apical periodontitis were recruited from
the endodontic clinic. Mandibular cuspids (n 2),
bicuspids (n 11), and molars (mesial roots) (n
27) were selected for the study. Bacterial sampling
was performed upon access and after each of two
consecutive instrumentations. The first instrumen-
tation utilized 1% NaOCl and 0.04 taper ProFile
rotary files. The cuspid and bicuspid canals were
instrumented to a #8 size and the molar canals to a
#7 size. The second instrumentation utilized Light-
Speed files and 1% NaOCl irrigation for further
enlargement of the apical third. Typically, molars
were instrumented to size 60 and cuspid/bicuspid
canals to size 80.
Our findings show that 100% of the cuspid/bi-
cuspid canals and 81.5% of the molar canals were
rendered bacteria-free after the first instrumenta-
tion sizes. The molar results improved to 89% after
the second instrumentation. Of the (59.3%) molar
mesial canals without a clinically detectable com-
munication, 93% were rendered bacteria-free with
the first instrumentation.
Using a Wilcoxon rank sum test, statistically sig-
nificant differences (p < 0.0001) were found be-
tween the initial sample and the samples after the
first and second instrumentations. The differences
between the samples that followed the two instru-
mentation regimens were not significant (p
0.0617). It is concluded that simple root canal sys-
tems (without multiple canal communications) may
be rendered bacteria-free when preparation of this
type is utilized.
779
Printed in U.S.A.
VOL. 28, NO. 11, NOVEMBER 2002
The endodontic literature has clearly established the role of bac-
teria and their byproducts in the production of apical periodontitis,
whereas scientific evidence for nonmicrobial causes of apical
periodontitis is lacking (1 4). Once infected, the root canal space
seems to act as a selective incubation chamber for bacteria (5). An
improved prognosis is seen when no cultivatable bacteria are found
at the time of obturation (6 8) and when no apical lesion is present
(9 11). Therefore, the main goal of endodontic treatment is to
prevent or eliminate infection within the root canal system.
Instrumentation has long been considered the cornerstone of
endodontic treatment, yet only a few studies have focused on its
antibacterial effects.
Bystrom and Sundqvist (12) cultured 15 necrotic teeth after
instrumentation with saline irrigation. They found a 100- to 1000-
fold reduction in the bacterial counts, yet no teeth cultured bacte-
ria-free after the first appointment. The addition of irrigating so-
lutions further improved bacterial elimination but could not
predictably remove all bacteria. Other studies by rstavik et al.
(13) and Matsumiya and Kitamura (14) suggested that the size of
apical instrumentation may be important for the effective removal
of canal bacteria. These studies found that with larger instrumen-
tation fewer bacteria remained in the canal and the healing was
more rapid. More recently, Wu and Wesselink (15) confirmed that
molar canals were much cleaner when instrumention reached a size
#45 apical file.
The removal of bacteria by NiTi instrumentation was evaluated
in two recent studies by Dalton et al. (16) and Shuping et al. (17).
The results from Dalton et al. (16) mirrored those of Bystrom and
Sundqvist (12). They found significant bacterial reduction with
instrumentation and saline irrigation, but only 28% of the teeth
sampled free of bacteria. Also, no significant difference was found
between the instrumentation with either stainless steel hand files or
rotary NiTi instrumentation. The addition of NaOCl in the Shuping
et al. study resulted in a better antibacterial effect but only after the
instrumentation exceeded ISO size #30 35 (17). It was found that
NaOCl provided no benefit over saline with the smaller instru-
mentation sizes. Both studies demonstrated improved canal disin-
fection with progressively larger instrumentation, but instrumen-
tation was again unable to predictably remove all canal bacteria.
The need for large apical preparations may be explained by the
study by Kerekes and Tronstad (18). They showed that 95% of the
molar mesial canals that they evaluated would have required at
least a size #60 apical preparation to fully instrument the apical 1
mm. They also showed that 65% of the mesial canals communi-
780 Card et al.
cated. This finding points to one potential problem with the Dalton
et al. (16) and Shuping et al. (17) studies, because they instru-
mented and sampled only the molar mesiobuccal canals and failed
to account for the expected communication between the canals.
The purpose of this study was to evaluate whether a larger apical
instrumentation than used in the Dalton et al. (16) and Shuping et
al. (17) studies would improve bacterial removal and to determine
if the combined instrumentation of both mesial canals (in mandib-
ular molars) would contribute to a further reduction in positive
cultures.
MATERIALS AND METHODS
Subject Recruitment and Qualification
Approval for this project was obtained from the University of
North Carolina School of Dentistrys Committee on Investigation
Involving Human Subjects. The primary investigator (S.C.) con-
ducted all clinical and sampling procedures. The study subjects
presented to the endodontic clinic at the University of North
Carolina School of Dentistry for evaluation and treatment of apical
periodontitis. The qualified subjects were accepted into the study
on a nonrandom, consecutive basis.
Only mandibular canines, first and second premolars, and first
and second molars were included in the study. To qualify, the teeth
must have had radiographic evidence of a periapical radiolucent
lesion associated with the tooth, no response to thermal or electric
pulp testing, enough crown structure for adequate isolation, and no
history of previous endodontic treatment of the tooth. Additionally,
the canines and premolars needed to have a single canal. Two
mandibular cuspids, 11 bicuspids, and 27 molars were selected for
the test teeth. An additional two bicuspids and three molars served
as controls.
Instrumentation and Bacterial Sampling
After explanation of the study and patient consent, the patient
was anesthetized and the tooth was isolated with a rubber dam.
Dental floss was securely tied around the neck of the tooth, and
then the tooth and adjacent rubber dam were cleaned with 30%
hydrogen peroxide until no further bubbling of the peroxide oc-
curred. If difficulty occurred in attaining a bubble-free status,
Oraseal Putty (Ultradent Products Inc., South Jordan, UT) was
placed around the neck of the tooth and the process repeated. All
surfaces were then coated with tincture of iodine and allowed to
dry. The method for molar treatment varied slightly from the
method for the single-rooted teeth due to the need to block off the
distal canal in the molar before sampling and the need to instru-
ment both mesial roots during treatment. Only the mesiobuccal
canal of the first and second molars was sampled, but both me-
siobuccal and mesiolingual canals were fully instrumented. Sterile
orifice openers (Tulsa Dental Products, Tulsa, OK) were used to
open the orifice of the distal canal before sealing the distal canal
orifice with Cavit (ESPE, Norristown, PA). These files were set
aside and another set of sterile orifice openers was used to initiate
access into the two mesial canals. Sterile saline was again used to
flush any debris from the chamber. The chamber was then dried
with sterile cotton pellets and/or paper points, and 0.02 ml of dental
transport fluid (DTF) (Anaerobe Systems, Morgan Hill, CA) was
placed into the sampled mesiobuccal or single canal with a sterile
Journal of Endodontics
tuberculin syringe. The canal contents were then dispersed with
#10 20 size K-files placed to within 1 mm of the estimated
working length and transferred to the DTF bottle, with sterile
xxfine-to-fine paper points (Mynol, Block Drug Corp, Jersey
City, NJ) placed as close to working length as possible. This
constituted the first sample (S1).
Working length was established within 1 mm of the apex and
confirmed radiographically. The canals were then instrumented
with the 0.04 ProFile tapers (ProFile 0.04 TapersTM Series 29TM,
Tulsa Dental Products) to the predetermined final file size. For the
mesial canals of molars this was a #7 (0.465 mm) size ProFile, and
for single-rooted teeth it was a #8 (0.599 mm) size ProFile. Due to
severe canal curvature, one molar was instrumented to a #6 (0.360)
size ProFile. No other exceptions occurred. All ProFile rotary
instrumentation utilized a Micro Mega MM324 air motor with a
model MM10TE contra-angle (1:6 reduction) attachment (Tulsa
Dental Products) rotating at 333 rpm. If a file would not go to
length, the next larger size file was used to remove more coronal
dentin and facilitate reaching the desired length.
The canals were categorized as having a clinically detectable
communication if the final Profile used, when placed to length in
the mesiobuccal canal, prevented another Profile placed in the
mesiolingual canal from reaching the prepared length, or
vice-versa.
Final instrumentation was performed with LightSpeed rotary
instruments and a portable handpiece rotating at 2000 rpm (Light-
Speed Technology Inc., San Antonio, Texas). Instrumentation pro-
gressed to the size requiring 7 to 10 pecks to reach working length,
as recommended by the manufacturer. The molar sizes ranged
from 57.5 to 65, and the bicuspid/cuspid canals ranged from 80 to
100 [Fig. 1 (AC)]. All teeth were copiously irrigated with 1%
NaOCl after each successive instrument, using a 28-gauge Double
D needle (Beutlich Pharmaceuticals LP, Waukegan, IL) placed as
far apically as possible without wedging.
The sampling technique used for the S2 and S3 samples was the
PMR method developed by Moller (19), with the exception of
using Dental Transport Fluid (DTF) instead of VGMA II. The
DTF has the clinical advantage of being a liquid instead of a gel,
yet it still preserves the viability of the bacteria for 72 h. After each
instrumentation regimen, the canal was flushed with 2 ml of 5%
sodium thiosulfate to neutralize the NaOCl, rinsed with sterile
saline, and dried with sterile paper points. Approximately 0.02 ml
of DTF was placed into the canal with a sterile tuberculin syringe.
A sterile file, identical to the final file, was placed to length in the
canal and pumped five times with minimal reaming motion. The
canal contents were then soaked up with sterile paper points and
transferred to the DTF vial and labeled appropriately as either the
second Profile sample (S2) or third LightSpeed sample (S3).
Treatment of the remaining (molar) canals was completed, cal-
cium hydroxide was placed in all canals with a Lentulo spiral
filler (Caulk; Milford, DE), and the tooth was temporized with
intermediate restorative material (IRM) (Dentsply Int. Inc., Mil-
ford, DE). After a minimum of 7 days, the patient returned to the
clinic for completion of the treatment. This included anesthesia,
rubber dam isolation, flushing the canals with NaOCl, drying with
paper points, and obturation of the canals. In those canals instru-
mented with LightSpeed, the Simplifill system (LightSpeed
Technology Inc.) of obturation was used followed by backfilling
with warm gutta-percha (ObturaTM II, Fenton, MO).
Five teeth presented with radiographic and clinical signs of
apical periodontitis but were found to contain various amounts of
vital tissue in the canal and upon bacterial sampling proved to be
Vol. 28, No. 11, November 2002
FIG. 1. (A) Tooth #30. Mesial canals instrumented to size 65 and
distal canal to size 80 LightSpeed. (B) Tooth #20. Instrumented to
size 90 LightSpeed. (C) Tooth #31. Mesial canals instrumented to
size 70 LightSpeed. Distal canal instrumented to a size 60 K-file.
free of bacteria in the initial sample. These served as a negative
control to confirm the effectiveness of the method.
The bacteriological samples were immediately transferred to the
University of North Carolina Dental Microbiology Laboratory (a
Apical Enlargement and Bacterial Reduction 781
CLIA certified laboratory), and the lab personnel performed all
microbiological procedures. The samples were dispersed with a
vortex for 30 s before aliquot disbursement. A model D spiral
plater was used to plate nonselective and selective plates. This
device conveniently delivers 49 l of sample while accomplishing
a 2.3 log dilution across the plate. The plates were then incubated
for 1 to 3 days aerobically and 5 to 7 days anaerobically in an
anaerobic gas consisting of 5% CO2, 85% N2, and 10% H2. Growth
on the plates was directly counted using the grids designed for
the spiral plating system, and actual counts were calculated
based on the known dilution factors. Additionally, growth on
the agar plates was subcultured for identification on the basis of
anaerobic, facultative anaerobic, or aerobic growth. Gram stain,
micromorphology, colony morphology, selective growth media,
and standard (NCCLS) biochemical identification techniques
were utilized to aid in the identification of aerobic and anaer-
obic strains.
Statistical Analysis
A sign test was performed to determine if the differences be-
tween the three samples (S1-S2, S1-S3, and S2-S3) were signifi-
cant. It was also of interest to compare the molar data from this
study to the data from equivalently instrumented molars in the
Shuping et al. (17) study. A Wilcoxon rank sum test was used to
compare the results and determine statistical significance. To de-
termine the significance of canal joining, a 3 2 table was
constructed to compare the results from Shupings data to this
study, when canal joining was and was not found. Fischers exact
test was used due to low cell counts. A p-value of less than 0.05
was considered significant.
RESULTS
Bacteria were found initially in 38 of 40 test teeth and in no
control teeth. The mean count in the initial sample (S1) was 6.0
107, with a range between zero and more than could be counted
(109). A highly significant difference was observed between S1
and S2 (p 0.0001) and between S1 and S3 (p 0.0001). The
difference between S2 and S3 was not significant (p 0.0617).
However, the S2 and S3 comparison was made using the data
from only the five subjects that were found to have culturable
bacteria after the first instrumentation regimen (at S2). Addi-
tionally, the bacteria found in the positive second and third
cultures were found to be components of the previous sample(s)
from that canal.
All (13/13) of the test bicuspids and cuspids cultured bacteria-
free after both the first and second instrumentations. Of the 59.3%
(16/27) molar mesial canals without a clinically detectable com-
munication between the mesiobuccal and mesiolingual canals,
93% (15/16) cultured bacteria-free after both the first and second
instrumentations. Overall, 81.5% (22/27) of the molar canals cul-
tured bacteria-free after the first instrumentation (S2) and 88.9%
(24/27) after the second instrumentation (S3). When the nonjoining
canals in this study were compared to the data from Shuping et al.,
no significant difference was found (p 0.0952). All five of the
control samples showed no bacterial growth in any of the three
samples.
782 Card et al.
DISCUSSION
A strong correlation between the presence of bacteria in a root
canal and apical periodontitis was confirmed in this study, because
95% (38/40) of the test teeth harbored bacteria in the first sample
(S1). These findings compare favorably to the findings of
Sundqvist (2), rstavik et al. (13), Dalton et al. (16), and Shuping
et al. (17), who found 96% to 98% of their necrotic samples to
contain culturable bacteria.
Of the 21 molars in the Shuping et al. study (17) that were
instrumented to sizes equal to those used in this study, 71.4%
(15/21) produced bacteria-free cultures. Of those instrumented to
smaller sizes, only 44% (4/9) cultured bacteria-free after instru-
mentation. Although Shuping et al. did not account for the joining
of mesial root canals, there was improved bacterial reduction with
increased instrumentation sizes. This study supports their findings.
Of the 27 molars instrumented to size #7 (0.465 mm), 22 (81.5%)
did not have culturable bacteria after instrumentation and irriga-
tion. These results improved to 24 (88.9%) after further apical
enlargement with the LightSpeed to sizes in excess of 0.575 mm.
One concern with the Dalton et al. (16) and Shuping et al. (17)
molar samples was the potential for positive culture results due to
the joining of the uninstrumented mesiolingual canal with the
sampled mesiobuccal canal. This was a likely problem, because
Kerekes and Tronstad (18) found approximately 65% of mandib-
ular molars have a direct communication. Although it is likely
impossible to detect all canal communications clinically, an at-
tempt was made to evaluate this problem. With our relatively crude
criterion, we found that 40.7% of the molar mesial canals com-
municated, and based on Kerekes and Tronstads work, it is likely
that more communications were not discovered.
When the results from this study were further differentiated
between those canals that did not communicate and those that did,
it became obvious that canal communication was an important
clinical factor. Of the 15 molar canals without clinically detected
communication, 14 (93.3%) cultured bacteria-free at the S2 sam-
ple. In contrast, only four of seven (63.6%) of canals with com-
munication cultured bacteria-free. This result was not found to be
significant (p 0.0952), but might have been if all canal com-
munications could have been detected, parametric statistical anal-
yses had been possible, and more than five cases were involved in
the analysis.
The lack of significance between the nonjoining molar canals in
this study and the similarly instrumented canals from the Shuping
study may likely reflect the relatively small number of cases
involved, the unknown number of canals communicating in their
samples, and other minor differences that existed between the
studies.
There is a legitimate concern regarding the potential fracturing
of teeth instrumented to the sizes used in this study. A tapered file
with the same apical sizes as the LightSpeed files used in this
study might have destroyed many roots. The advantage of utilizing
the LightSpeed instruments is that cervical weakening is mini-
mized, because the short cutting edge does not remove cervical
root structure during the apical preparation. This may help to
alleviate concerns about the weakening of the tooth. However, no
studies to date have determined the safe limits of apical
instrumentation.
There is growing concern regarding the continued effectiveness
of chemical means of bacterial removal; and the developing resis-
tance of bacteria to chemical agents has become a hot topic of
discussion. Molander et al. (20) suggested that enterococci might
Journal of Endodontics
be resistant to the effects of calcium hydroxide, yet they account
for 32.4% to 47% of the culturable bacteria obtained from failed
root canal treatments. In this study, 13 of the 14 molars (93.33%)
without canal communication and all (100%) of the single canal
cuspids and bicuspids were found to have bacterial reduction that
equaled or surpassed that found in the Shuping et al. study when
a medication was used. If it is possible to predictably obtain the
bacterial removal found with this study, it may be advantageous to
utilize this technique to avoid the potential development of resis-
tant bacteria.
When the removal of bacteria is not as predictable, as evidenced
by the poorer results with joining canals, then medications should
be used. The reliance on medications for fewer cases may thereby
limit the potential for developing bacterial resistance and yet
maintain the effectiveness of the medication for when it is abso-
lutely necessary.
Although there seems to be evidence supporting an improved
long-term prognosis when a negative culture is obtained, this study
was not designed to evaluate the success of treatment but merely
the success in obtaining a negative culture. Certainly our results are
encouraging, but long-term studies comparing the efficacy of this
type of treatment to standard regimens are required. Other teeth
will need to be evaluated, because it is likely that certain teeth will
have complex root canal anatomy and be poor candidates for this
type of treatment, whereas others will be easily treated.
It is concluded that a high percentage of the infected root canals
from mandibular cuspids, bicuspids, and molar mesial roots will no
longer harbor cultivatable bacteria when instrumented to the sizes
used in this study and that with many teeth this regimen may
substitute for treatment by a two-stage procedure utilizing an
intracanal dressing.
This study was funded in part by a grant from Steven Senia of LightSpeed
Technology Inc., San Antonio, Texas.
Dr. Card is a third-year graduate student; Dr. Sigurdsson is assistant
professor and graduate program director; and Dr. Trope is the J. B. Freedland
Professor and Chair, Department of Endodontics, The University of North
Carolina, Chapel Hill, North Carolina. Dr. rstavik is senior scientist, Scandi-
navian Institute of Dental Materials, Haslum, Norway. Address requests for
reprints to Dr. Martin Trope, Department of Endodontics, The University of
North Carolina, School of Dentistry, CB#7450, Chapel Hill, NC 27599.
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