Basic Research—Technology
The Influence of Preparation Size on the Mechanical
Efficacy of Root Canal Irrigation In Vitro
Kenneth W. Falk, DDS, and Christine M. Sedgley, PhD
Abstract
This study tested the hypothesis that the mechanical
efficacy of irrigation in reducing intracanal bacteria is
dependent on canal preparation size. Root canals of 30
extracted permanent cuspids were prepared consecu-
tively to sizes 36, 60, and 77 at working length (WL)
using ProFile 0.04 taper Series-29 files. Smear layer was
removed and teeth autoclaved following completion of
each instrumentation sequence. Root canals were in-
oculated with a 15-␮l suspension (1 ⫻ 106 cells) of
Pseudomonas fluorescens 5RL, a salicylate-inducible
bioluminescent reporter strain. Bioluminescence was
measured before inoculation (background), after inoc-
ulation and after irrigation with 6 ml of irrigant deliv-
ered 1 mm from WL using a 28G safety-ended needle.
The percentage of bacteria remaining following irriga-
tion was 26.95 ⫾ 9.71%, 10.46 ⫾ 5.87%, and 10.64 ⫾
6.01% for sizes 36, 60, and 77, respectively (p ⬍ 0.001;
repeated-measures ANOVA), with no difference be-
tween sizes 60 and 77 (p ⬎ 0.05; Tukeys). Irrigation 1
mm from WL was significantly less effective in canals
prepared to size 36.
Key Words
Bacteria, bioluminescence, endodontic, irrigation, prep-
aration size
From the Department of Cariology, Restorative Sciences
and Endodontics, University of Michigan Dental School, Ann
Arbor, Michigan.
Address requests for reprint to Dr Christine Sedgley, De-
partment of Cariology, Restorative Sciences and Endodontics,
University of Michigan, School of Dentistry, 1011 N. University
Drive, Ann Arbor, MI 48109-1078. E-mail address: csedgley@
umich.edu.
Copyright © 2005 by the American Association of
Endodontists
742 Falk and Sedgley
I n 1859 Jonathon Taft advocated “frequent syringing” of root canals to remove “the
offensive gasses or fluids of decomposition” (1). However, the specific removal of
microorganisms from root canals became a goal of root canal treatment only after the
publication of several seminal papers by Willoughby Dayton Miller throughout the
1890s (2). Nonetheless, despite more than a century of technological improvements in
root canal instrumentation procedures, clinical studies indicate that bacteria remain in
the canal following standardized cleaning and shaping procedures using nonantimicro-
bial irrigants (3, 4). Additionally, the use of antimicrobial irrigants have been shown to
further reduce bacteria but without complete elimination (5– 8).
In vitro studies have shown that increased apical enlargement aids mechanical
debridement of particles (9) and debris detected histologically (10, 11). In addition, a
recent clinical study demonstrated that microbial debridement, accompanied by irri-
gation with 1% sodium hypochlorite (NaOCl), was more effective with increased apical
enlargement (8). The authors found no bacteria remaining in any of the cuspid and
bicuspid canals prepared to size 60, however, 81.5% of molar canals instrumented to
size 46 had residual bacteria. When the molars were prepared to apical size 60, 89% of
the canals were bacteria-free.
While clinical investigations are preferred, an advantage to in vitro experimenta-
tion is the ability to control variables. For example, in the aforementioned clinical
studies the irrigant volume and needle depth penetration varied. In addition, the most
common method of evaluating root canal bacteria assesses growth from paper point
samples (8), in effect measuring what is removed from the root canal rather than what
remains behind. This obstacle was addressed using an in vitro method that measured
radioactively labeled bacteria remaining in instrumented canals (12); however, the
protocol required destruction of the tooth sample to obtain a reading, thereby render-
ing impossible the assessment of sequential treatment procedures. Recently, a nonde-
structive in vitro method utilizing the bioluminescent reporter strain Pseudomonas
fluorescens 5RL was described that can assess sequential instrumentation treatment
procedures and quantification of bacteria within the canal without destroying the sam-
ple (13). Using this method it was shown that irrigation efficacy was significantly im-
proved using 6 ml compared to 3 ml of irrigant (13) and when delivered 1 mm
compared to 5 mm from working length (WL) (14). However, both studies used teeth
with canals prepared to size 60.
The hypothesis tested in this study was that the mechanical efficacy of irrigation in
the removal of intracanal bacteria is dependent on the canal preparation size. Biolu-
minescent bacteria were inoculated into sequentially enlarged root canals of the same
teeth, after which canals were irrigated with a nonantimicrobial irrigant and remaining
bacteria quantified using real-time bioluminescent imaging (BLI).
Materials and Methods
Teeth
Thirty permanent cuspids with straight roots, clinically intact crowns, no restora-
tions, no cracks, no caries and of approximate length 24 mm were selected from teeth
stored in 50% glycerine/50% ethyl alcohol at the University of Michigan School of
Dentistry (Ann Arbor, MI). Teeth were radiographed from both mesial and buccal views
to assess canal morphology and those identified as having abnormal anatomy or calci-
fied root canal systems were excluded. Teeth were autoclaved at 121°C for 15 min at 26
psi and stored at 25°C in 100% humidity until use.
JOE — Volume 31, Number 10, October 2005
 Basic Research—Technology
Coronal access openings were prepared using tungsten carbide TABLE 1. Influence of preparation size on the mechanical efficacy of irrigation
burs in a high-speed handpiece with constant water spray. Root canal Canal preparation size
WL was set at 1 mm short of the apical foramen based on visual inspec-
tion of a size 10 stainless steel K-file extending beyond the apical fora- 60 77
36
men. Canals were instrumented using Orifice openers and Profile .04 (p value) (p value)
Series 29 Rotary Ni-Ti files (Dentsply Tulsa Dental Products, Tulsa, OK). Log10
Orifice openers were used to prepare the coronal one-third of canals reduction
Mean 0.59 1.03 1.06
and the Profile .04 Series 29 were used to prepare the middle and apical SD 0.16 0.25 0.31
two-thirds of canals. Overall (⬍0.0001†)
36 — (⬍0.001‡) (⬍0.001‡)
Preparation of Teeth for Sequences 1 (size 36), 2 (size 60), 60 — — (⬎0.05‡)
and 3 (size 77)
The 30 teeth were instrumented to final apical size 36 for the first Percentage of bacteria remaining in root canal (%)
Mean 26.95 10.46 10.64
experiment (sequence 1). Copious irrigation with 5.25% sodium hypo- SD 9.71 5.87 6.01
chlorite was used between instruments. After instrumentation, the
smear layer was removed from canal walls by ultrasonic treatment for 4 Absolute numbers of bacteria remaining in root canal
min each in 17% EDTA and 5.25% NaOCl (15). Canals were then rinsed Mean 2.7.E ⫹ 05 1.0.E ⫹ 05 1.1.E ⫹ 05
with copious sterile distilled water and ultrasonically treated for 4 min SD 9.7.E ⫹ 04 5.9.E ⫹ 04 6.0.E ⫹ 04
with distilled water after which the apices were coated with a clear nail * 1⫻106 bacteria inoculated into root canals (n ⫽ 30) for each experiment.
varnish to seal the apical foramen. Teeth were then autoclaved at 121°C † Repeated measures ANOVA, p ⬍ 0.0001.
for 15 min at 26 psi and stored aseptically in 100% humidity at 25°C ‡ Tukey’s multiple comparisons.
until use. Canals were dried with sterile paper points (Kerr USA, Romu-
lus, MI) immediately before each experiment.
The above procedures were repeated on the same 30 teeth for sizes system (Xenogen Corp., Alameda, CA) coupled to a data-acquisition PC
60 (sequence 2) and 77 (sequence 3). Teeth were autoclaved at the running LivingImage software (Xenogen Corp.). The imaging system
completion of each sequence. includes a highly sensitive charge coupled device (CCD) camera, a dark
imaging chamber to minimize incident light, and software to quantify
Bacteria the results. The CCD camera can detect very small numbers of photons
All investigations used P. fluorescens 5RL, a salicylate inducible as well as operate as a traditional camera and create real-time images
bioluminescent reporter strain harboring the plasmid pUTK21, a nah with extremely low noise.
sal-lux reporter plasmid (approximate size 120kb) that contains a Teeth were positioned in the dark imaging chamber at 25°C. A gray
salicylate-inducible luxCDABE gene cassette from Vibrio fischeri (16, scale tooth surface image was collected in the chamber under dim
17). Bioluminescent bacteria cultures were prepared from -80°C fro- illumination, followed by acquisition and overlay of the pseudocolor
zen stocks in 300 ml Erlenmeyer flasks containing 100 ml LB broth image representing the spatial distribution of detected photon counts
(Difco; Becton, Dickinson and Company, Sparks, MD) with salicylate emerging from active luciferase within the tooth. An integration time of
(50 ␮g/ml) (LBS) in aerobic conditions at 25°C with shaking. After 1 min was used for acquisition of the luminescent image (photons/sec/
24 h, a subculture was prepared and grown to OD600 0.190 measured cm2/sr). Signal intensity was quantified as the sum of all detectable
using a spectrophotometer (Spectronic 20D⫹, Thermo Electron Cor- photon counts (photons/sec) within a reproducible region of interest
poration, Pittsburgh, PA) with corresponding bioluminescence of prescribed over the entire tooth.
7.5 ⫻ 105 RLU measured using a single tube luminometer (Sirius-2, Images and photon counts were obtained before inoculation of
Zylux Corp., Oak Ridge, TN). bacteria (background), after inoculation of bacteria, and after 6 ml of
Viable cell counts were determined using 10-fold serial dilutions irrigation.
prepared in nuclease-free water supplemented with salicylate (50 ␮g/
ml) (H2O/Sal). Viable counts were obtained for each experimental se- Statistical Analysis
quence by plating serial dilutions in triplicate on LBS agar, and counting Photons from bioluminescent bacteria in each tooth were ob-
colony forming units after 48 h aerobic incubation at 25°C. tained by subtracting background photon counts. A log10 transforma-
Undiluted bacterial suspension in LBS (15 ␮l) was pipetted into tion of bacterial photons was performed because of the extensive range
each root canal using fine sterile gel-loading pipets (Fisherbrand, of photon readings. The data were confirmed to have normal distribu-
Fisher Scientific, Pittsburgh, PA) as previously described (13). Pipet tip tion on frequency histograms. Repeated measures ANOVA and Tukey’s
placement for inoculation of bacteria was 5 mm from the WL for each multiple comparisons were performed on log10 transformed data using
sequence. Prism 4 for Macintosh software (GraphPad Software, Inc. San Diego,
CA). Significance was set at p ⬍ 0.05.
Irrigation
A total of 6 ml of irrigation with sterile distilled water/salicylate was Results
delivered via a sterile 28 gauge safety-ended endodontic irrigating nee- Viable counts obtained by culturing determined that approxi-
dle (Max-I Probe, Dentsply Limited, Waybridge, Surrey, England) mately 1 ⫻ 106 bacterial cells were inoculated into the canals. Real-
placed 1 mm short of WL as previously described (13). time BLI data showed that the mean percentage bioluminescent bacteria
remaining following irrigation was 26.95 ⫾ 9.71, 10.46 ⫾ 5.87, and
Measurement of Bioluminescence in Teeth 10.64 ⫾ 6.01 for sizes 36, 60, and 77, respectively, corresponding to a
BLI was performed at the Michigan Small Animal Imaging resource mean log10 decrease of 0.59 ⫾ 0.16, 1.03 ⫾ 0.25, and 1.06 ⫾ 0.31,
(Department of Radiology, University of Michigan) as previously de- respectively (p ⬍ 0.001; repeated-measures ANOVA) (Table 1). There
scribed (13). Imaging was conducted using a cryogenically cooled IVIS was no difference between sizes 60 and 77 (p ⬎ 0.05; Tukey’s multiple

JOE — Volume 31, Number 10, October 2005 Preparation Size Influencing Efficacy of Canal Irrigation 743
Basic Research—Technology
Figure 1. Images of representative tooth. (A) Background, no bacteria, (B) 1 ⫻
106 P. fluorescens 5RL in root canal. NB. Bacterial cell count was determined
by culturing the same volume of inoculum. (C–E) After 6 ml irrigation delivered
using 28 gauge Max-I-Probe needle positioned 1 mm from WL after root canal
instrumented to (C) size 36, (D) size 60, and (E) size 77. Color bar on right
gives bioluminescence image units (photons/sec/cm2/sr).
comparisons). A series of bioluminescent images over all sequences for
a representative tooth is shown in Fig. 1.
Discussion
The need for adequate enlargement of the root canal to allow
irrigation was recognized by Grossman in 1943 (18). The results of this
study show that canal preparation size significantly influences the me-
chanical efficacy of irrigation. Compared to preparation sizes 60 and 77,
irrigation was significantly less effective in canals prepared to size 36.
From a starting inoculum of 1 ⫻ 106 cells, approximately 3 ⫻ 105
viable cells remained in canals instrumented to size 36 following 6 ml
irrigation delivered 1 mm from WL. Increasing canal enlargement to
size 60 resulted in significantly less bacteria remaining in the root canal
system. These latter findings are essentially in agreement with others (8,
12). The debridement of root canals infected with radiolabeled Entero-
coccus faecalis was more effective when canals were instrumented to
size 50 using Pow-R instruments compared to size 35 using GT and
Profile instruments (12) and apical enlargement resulted in less cul-
turable bacteria being recovered clinically (8).
Interestingly, in the present study there was no difference in the
number of remaining bacteria in canals prepared to sizes 60 and 77,
indicating that enlargement beyond size 60 did not offer an advantage in
terms of irrigation efficacy. In addition, approximately 10% of bacterial
cells still remained in these canals after irrigation. These latter data
support clinical reports that complete removal of all bacteria is not
predictable in canals instrumented at least four file sizes larger than size
40 despite irrigation with 0.5% or 5% NaOCl (5, 19). In contrast, Card
et al. (8) reported no culturable bacteria when bicuspid canals were
instrumented to size 60 using 1% NaOCl. These seemingly contradictory
reports of presence and absence of bacteria could be explained in part
by the greater detection sensitivity of the present method. Other vari-
ables found in clinical studies also could contribute, such as apical
placement of needle tip, irrigation needle gauge and volume of irrigant.
For example, Card et al. (8) “copiously irrigated” canals using a needle
placed as far apically as possible without binding. An important consid-
eration in clinical studies is the feasibility of standardizing all clinical
procedures. The opportunity existed in this in vitro study to control
needle depth and irrigant volume.
Previous methods to assess the effect of instrumentation size on
irrigation efficacy have used culture assessment of paper points, mea-
surement of remaining radioactively labeled bacteria (12) and histo-
logical observations of debris remaining on canal walls (10, 11). De-
termining efficacy of chemomechanical procedures by culturing does
not allow evaluation of bacteria remaining within the canal, while his-
tological methods are essentially qualitative in nature. The present
method used real time BLI that allowed quantification and sequential
744 Falk and Sedgley
analysis; photon emissions from bacteria inoculated in root canals can
be detected using real-time BLI (13). More importantly, by using each
tooth sample as its own control throughout sequential procedures, the
potential impact of variations between teeth was eliminated. In these
investigations, no attempt was made to “grow” bacteria in root canals,
nor to “kill” the inoculated bacteria, hence a nonantimicrobial irrigant
was used. Naturally occurring bioluminescent species would not be
expected in the infected root canal flora. Rather, the genetically engi-
neered strain P. fluorescens 5RL, a gram-negative rod 2 to 3 ␮m in
length, was selected for these investigations based on strong biolumi-
nescent activity associated with its reporter plasmid pUTK21 when
grown in the presence of salicylate (17).
A consideration, when designing a study that used the same teeth
over a number of progressive sequences, was whether the teeth would
remain intact throughout repeated cycles of autoclaving and ultrasonic
treatment (for smear layer removal). Intentionally, only intact, caries-
free and unrestored teeth were used, and the teeth were kept in 100%
humidity when not being used in experiments. At the completion of
experiments, all the teeth remained intact. This indicates that a greater
number of sequential procedures are feasible. Future studies could use
a greater range of preparation sizes, including size 46 that was not
investigated here, as well as smaller apical sizes used in greater taper
instrumentation systems. An antimicrobial irrigant such as NaOCl,
which would be expected to further reduce the numbers of viable bac-
teria in root canals (5), also could be investigated in future studies using
this method.
In addition to canal size, other factors play an important role in
root canal debridement. For example, the influence of canal taper on
efficacy of debris removal was investigated in recent histological studies
(10, 11). Debris was more effectively removed using .04, .06, and .08
ProFile GT instruments when the apical preparation size was larger
(size 40 compared with size 20). However, when a taper of 0.10 was
produced at the apical extent of the canal, there was no difference in
debris removal between the two sizes (10). Factors shown to improve
the efficacy of root canal irrigation include closer proximity of the
irrigation needle to the apex (14, 20, 21), larger irrigation volume
(13), and narrower gauge irrigation needles (9). The present data
additionally show that the efficacy of irrigation is significantly reduced in
canals prepared to size 36 compared to size 60, but with no advantage
provided by further enlargement to size 77. Clinicians need to balance
the need for optimization of the mechanical efficacy of irrigation pro-
vided by enlargement of canals with the negative consequences of re-
duction in tooth structure and subsequent weakening of the root struc-
ture (22).
Acknowledgments
The authors gratefully acknowledge support from the Graduate
Endodontic Research Fund, University of Michigan.
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