Aust Endod J 2011; 37: 128–133

ORIGINAL RESEARCH

A comparison of three rotary systems and hand

instrumentation technique for the elimination of Enterococcus
faecalis from the root canal aej_239 128..133

Melahat Gorduysus, DDS, PhD ; Emre Nagas, DDS ; Ozgur Yildirim Torun, DDS, PhD2; and
1 1

Omer Gorduysus, DDS, PhD1

1 Department of Endodontics, Faculty of Dentistry, Hacettepe University, Ankara, Turkey
2 Department of Clinical Microbiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

Keywords
bacteria, hand instrumentation, HeroShaper,
K3, ProTaper, rotary instrument.
Correspondence
Dr Melahat Gorduysus, Department of
Endodontics, Faculty of Dentistry, Hacettepe
University, 06100 Sıhhiye, Ankara, Turkey.
Email: mgorduysus@yahoo.com
doi:10.1111/j.1747-4477.2010.00239.x
Abstract
The aim of this study was to compare the in vitro reduction of a bacterial
population in a root canal by mechanical instrumentation using three rotary
systems and hand instrumentation technique. The root canals contaminated
with a suspension of Enterococcus faecalis were instrumented using ProTaper, K3,
HeroShaper and K-file hand instrumentation technique. Later the root canals
were sampled. After serial dilutions, samples were incubated in culture media
for 24 h. Bacterial colonies were counted and the results were given as number
of colony-forming units per millilitre. The results showed that all the canal
instrumentation systems reduced the number of bacterial cells in the root
canals. Statistically, ProTaper instruments were more effective in reducing the
number of bacteria than the other rotary files or hand instruments.

support the treatment objectives (11). However, bacterial

Introduction
Microorganisms play a fundamental role in the aetiology
of pulp and periapical disease (1,2), and their control and
elimination are important during endodontic treatment
(3). Reducing the bacterial count in infected root canals is
accomplished by a combination of mechanical instru-
mentation, various irrigation solutions and antibacterial
medicaments placed into the canal.
Mechanical instrumentation is the core method
for bacterial reduction during endodontic treatment of
infected root canals (4). Irrigation and dressing with anti-
microbial agents further reduce or eliminate the number
of intracanal bacteria (5–7). The effect of canal instru-
mentation using non-antimicrobial irrigation is reported
in the previous studies (8,9). It has been demon-
strated that mechanical instrumentation with a non-
antimicrobial irrigant reduces the intracanal bacterial
count enough to detect a quantifiable difference using
appropriate sampling techniques (10).
Bacteria in the canal can penetrate the dentinal tubules.
Increasing the size of the apical preparation reduces the
bacterial count and yields cleaner canals, which may
penetration is deeper coronally and at mid-root levels than
in the apical part (12,13). As a result, root canals instru-
mented with greater taper files may contain fewer bacteria
than those instrumented with smaller taper files (14).
Siqueira et al. (15) compared conventional taper files
(0.02 mm/mm) with greater taper files (0.06), and found
no significant difference in bacterial reduction. Dalton
et al. (10) found no difference in bacterial reduction using
either 0.04 taper rotary or stainless-steel K-file hand
instrumentation with sterile saline as the irrigant. Sub-
stantial bacterial reduction was achieved with progressive
filing, regardless of file type and fewer bacteria remained
when larger file sizes were used. Neither of these tech-
niques could predictably render canals free of bacteria.
The ProTaper (Dentsply/Maillefer; Ballagigues, Switzer-
land), the K3 (SybronEndo; Orange, CA, USA) and
the HeroShaper (Micro-Mega; Besançon, France) have
become popular rotary systems, particularly among
general practitioners. Although the earlier developed
rotary nickel-titanium (Ni-Ti) systems (e.g. ProFile) have
published research evaluating their ability to remove
bacteria, there is only one currently published data in

128 © 2010 The Authors

Australian Endodontic Journal © 2010 Australian Society of Endodontology
M. Gorduysus et al.
peer-reviewed journals comparing the recently developed
rotary systems (ProTaper, HeroShaper) for ability to
remove bacteria (14).
The aim of this study was to compare the in vitro reduc-
tion of a bacterial population in root canals by mechanical
instrumentation using three rotary systems (ProTaper,
K3, HeroShaper) and K-file hand instrumentation.
Materials and methods
Selection and preparation of teeth
Experimental protocols were performed on freshly
extracted human lower premolars with a single-root
canal (n = 135). All experiments were conducted under
aseptic conditions in a laminar air flow cabinet to prevent
airborne bacterial contamination. Teeth were initially
stored in 10% formalin for 24 h, to provide disinfection
and organic tissue fixation. After this procedure, bone,
calculus and soft tissues on the root surface were
removed with curettes. The crowns were then sectioned
with a diamond disk and the root lengths were standar-
dised to 15 mm. They were then stored in physiological
saline solutions until use.
Canals were evaluated for apical patency with a size 10
file. Root canals were instrumented to a size 25 K-file
1 mm from the apex of the root (determined working
length is 14 mm) and irrigated with 2 mL of saline solu-
tion. The root canals were then filled with 17% ethylene
diamine tetraacetic acid for 3 min to remove the smear
layer. All the canals were finally rinsed with 5 mL of
distilled water. To prevent bacterial leakage, the apical
foramen was sealed with an epoxy resin and the surfaces
of all roots were varnished with nail polish. To improve
handling, the roots were mounted vertically in acrylic
blocks.
Sterilisation of specimens
All specimens were sterilised in an autoclave at 121°C for
15 min. The sterilised roots were randomly divided into
five groups with 27 teeth in each group and one of the
groups served as the positive control group. Before the
contamination procedure, a negative control group (total
of five roots) were collected by randomly selecting one
root from each group in order to confirm the sterilisation
procedure.
Contamination with Enterococcus faecalis
A pure culture of Enterococcus faecalis (ATCC 29212) was
used to contaminate the root canals. Colonies of E. faeca-
lis, grown on brain heart infusion (BHI, Difco; Detroit,
© 2010 The Authors
Australian Endodontic Journal © 2010 Australian Society of Endodontology
Elimination of Bacteria from the Root Canal
MI, USA) with 5% sheep blood agar plates incubated at
37°C for 48 h in a CO2 incubator (Sanyo MCO-18AIC,
Japan), were inoculated in BHI broth and left for 24 h.
A bacterial suspension was prepared in a fresh broth
from a culture of E. faecalis, grown in BHI broth for 24 h.
McFarland standard no. 4 was then used to evaluate the
broth in order to ensure that the bacterial count was
approximately 1.2 ¥ 109 colony-forming units per millili-
tre. Each root canal was completely filled with 20 mL of
E. faecalis suspension through their cervical apertures
using sterile automatic micropipettes. The root canals of
negative control were filled with 20 mL of sterile BHI
broth. The cervical access was sealed with temporary
cement (Nucavfil PSP Dental Co. Ltd. Belvedere, Kent,
UK). The roots were incubated at 37°C for 7 days with the
replacement of 20 mL of fresh BHI broth at the third day
after incubation.
Verification of specimen contamination
with E. faecalis
To confirm the contamination by E. faecalis, the negative
control roots were analysed by scanning electron micros-
copy (SEM) (JEOL JSM-6400, Japan). The specimens
were first fractured, fixed, completely dehydrated and
attached to slides. Then they were sputter-coated with
gold before SEM examination.
Root canal instrumentation
All procedures were carried out by the same researcher to
rule out personal variations.
Group 1: Twenty-five root canals were enlarged using
the ProTaper system (Dentsply/Maillefer; Ballagigues,
Switzerland). The ProTaper instruments were used in a
crown-down manner according to the manufacturer’s
instructions.
Group 2: Twenty-five root canals were enlarged using
the K3 system (Sybron Endo; Orange, CA, USA). The K3
instruments were used in a crown-down manner accord-
ing to the manufacturer’s instructions
Group 3: Twenty-five root canals were enlarged using
the HeroShaper system (Micro-Mega; Besançon, France).
The preparation was in a crown-down manner. The blue
HeroShaper sequence was employed.
Group 4: Twenty-five root canals were instrumented
using 0.02 tapered K-file hand instrument (Dentsply;
Maillefer; Ballagigues, Switzerland). The standard tech-
nique was used as described by Kerekes and Tronstad
(16). For coronal enlargement, #2, #3 and #4 Gates-
Glidden drills were used for the two-thirds of the coronal
part.
129
Elimination of Bacteria from the Root Canal
Group 5 (Control group): Twenty-five root canals
were used as the control group. In this group, no further
instrumentation or irrigation was done.
Each root canal was irrigated with a total volume of
10 mL sterile saline solution. Irrigation was delivered in
the canals using a 2.5 mL plastic syringe with a 23-gauge
needle.
Post-preparation specimen sampling method
Bacterial samples were acquired with #25 paper points.
The paper point was placed into the canal and kept there
for about 1 min and then transferred into tubes contain-
ing 1 mL saline for 1 min. After 10-fold serial dilutions
in saline, 100 mL aliquots were plated onto BHI agar
and incubated at 37°C for 24 h. Bacterial colonies were
counted and the results were expressed as the number of
colony-forming units per millilitre.
Statistical analysis
Data were expressed as mean ⫾ SEM. Kruskal–Wallis
variance analysis was used for determining the differ-
ences among groups and for multiple comparisons
Dunn’s post hoc test was used. A P-value less than 0.05
was considered statistically significant.
Results
In all of the specimens studied by SEM, the presence of
bacteria in the root canals as well as within the dentinal
tubules was demonstrated (Fig. 1).
Figure 1 Sample scanning electron microscopy picture of a root canal
contaminated with Enterococcus faecalis.
130
M. Gorduysus et al.
There was no bacterial growth in the negative control
roots.
All the canal instrumentation systems appreciably
reduced the number of bacterial cells in the root canals
(Fig. 2). Comparisons with the control group showed
that the ProTaper system was more effective (P < 0.001)
in reducing the number of bacteria than the K3 system
(P < 0.001), HeroShaper system (P < 0.001) and the
K-file hand instrumentation technique (P < 0.05). The
reduction of bacterial numbers was also significantly
greater in the ProTaper system when compared with the
K3 system (P < 0.05), HeroShaper system (P < 0.05) and
the K-file hand instrumentation technique (P < 0.001).
There were no other statistically significant differences.
Discussion
One of the most important objectives during root canal
instrumentation is the removal of vital and/or necrotic
pulp tissue, infected dentin and dentin debris in order to
eliminate most of the microorganisms from the root canal
system (17).
Several methods have been described to evaluate endo-
dontic instrumentation techniques, including microscopic
observation of the remaining debris, morphometric
analysis and a bacteriological assessment. A bacteriologi-
cal assessment was chosen for the present study because
of the importance of canal disinfection in successful treat-
ment (10).
Mechanical instrumentation is the core method for
bacterial reduction in the infected root canal (8,9). It is
Figure 2 The reduction of bacterial numbers in root canals by ProTaper,
K3, HeroShaper and K-file systems. CFU, colony-forming unit. All data are
expressed as mean ⫾ SEM. *P < 0.05, ***P < 0.001.
© 2010 The Authors
Australian Endodontic Journal © 2010 Australian Society of Endodontology
M. Gorduysus et al.
well established that mechanical preparation with saline
as the irrigating solution, cannot predictably eliminate
the bacteria from infected root canals. Possible bacterial
invasion into dentinal tubules and the lateral canals
from the main root canal, the complexity of the root
canal system in most teeth, the physical limitations of
instruments and the insignificant antibacterial activity of
saline may all play a part in bacterial regrowth (9).
Hence, the use of irrigation solutions is an important
part of effective chemomechanical preparation. As our
aim was to assess the effects of the instrumentation
techniques alone, an antibacterial irrigation solution
was not used in this study.
All techniques reduced the number of bacteria recov-
ered from infected canals (8,9,18). Standard instrumen-
tation with stainless-steel K-files was performed as a
comparative technique. However, other clinical effects of
large instrumentation, including compromised restor-
ability, fracture susceptibility and canal path alterations,
should also be considered when using any instrumenta-
tion technique (10). Lam et al. (19) showed that greater
apical enlargement (Lightspeed) or increased canal taper
(Greater Taper files) did not increase fracture suscep-
tibility of tooth roots.
NiTi rotary instrumentation is faster and its lower
rate of procedural errors has popularised its use (4). Such
features are biologically important if the practitioner
is to safely instrument to larger file sizes, thereby fur-
ther reducing intracanal bacteria. However, Rollison
et al. (20)showed that a standardised preparation with
increased apical enlargement of root canals appears to
result in significantly greater bacterial removal than
tapered instrumentation preparations. These findings are
in agreement with those of Ørstavik et al. (9) and Dalton
et al. (10). Although manual instrumentation is still the
most popular means of root canal preparation, many
specialists and an increasing number of general practitio-
ners are using rotary Ni-Ti instruments. However, rotary
instruments have not always been found to be superior to
hand instruments, when the various aspects of prepara-
tion were compared (21). Ahlquist et al. (22)and Schafer
and Lohmann (23)showed that hand instrumentation
produced cleaner canals than preparation with rotary
instruments.
E. faecalis was chosen as the bacteriological marker in
this study. It is a non-fastidious, easy-to-grow facultative
anaerobic bacterium of significant clinical importance
that can be used in a study applying a bacteriological
assessment method. Other bacteria commonly asso-
ciated with endodontic infections may require symbiotic
support from other bacteria, but E. faecalis has been
reported to survive and successfully increase alone (24).
The SEM evaluation used in the present study confirmed
© 2010 The Authors
Australian Endodontic Journal © 2010 Australian Society of Endodontology
Elimination of Bacteria from the Root Canal
Table 1 Mean of colony counts (CFU mL-1)
Control ProTaper K3 HeroShaper K-file
Mean 870 400 29 544 133 840 183 776 212 080
SEM 135 927 9 468.083 23 711.02 49 583.0405 32 803.35
SD 679 635.2 47 340.42 118 555.1 247 915.203 164 016.7
25 25 25 25 25
CFU, colony-forming unit.
that the specimens were successfully reinfected with the
organism (Fig. 1).
In this study, all three rotary systems were used with
the crown-down technique. Effective cleaning of root
canals may also be influenced by the apical size of a
preparation (25). In the present study, the apical pre-
paration was standardised in four techniques to a size
30.
It has been shown that the bacterial count is decreased
with larger instrument sizes (10). The bacterial numbers
continue to decrease with progressive filing to larger sized
files. This study showed that the ProTaper system was
more effective in reducing the number of bacteria than
the HeroShaper system, K3 and K-file hand instrumen-
tation technique (Table 1 and Fig. 2). It was expected that
the more aggressive removal of dentin (ProTaper) would
eliminate more bacteria and lead to lower bacterial counts
in the final test sample.
Siqueira et al. (15) compared conventional taper files
(0.02 mm/mm) with greater taper files (0.06), and found
no significant difference in bacterial reduction. In the
mentioned study, further apical instrumentation with the
conventional files to a larger size was significantly more
effective in eliminating bacteria from the root canal than
greater taper files that stopped at a smaller apical size,
supporting the concept that the size of the apical prepa-
ration is important in intracanal bacteria reduction
(9,11).
The reason for the differences in these studies could be
the test sample. Infection of the canals in vitro may have
affected the results (14), or the anatomy of the root canals
may have been a contributory factor. Mandibular premo-
lars, in horizontal section, have an oval-shaped root
canal, which is broad in the buccolingual direction
until the middle-third of the root canal (26). Surfaces
untouched by the instruments may remain.
In this study, the bacteriological sampling was accom-
plished with a sterile paper point to observe the root canal
contents. The paper point was then transferred to tubes
containing saline solution. The use of paper points has
the advantage that it can be performed in vitro and in vivo.
On the other hand, bacteriological sampling with paper
points is limited because only the microorganisms that
131
Elimination of Bacteria from the Root Canal
are in the root canal can be sampled, while those located
inside the dentin tubules can not (21).
It is suggested that further studies be performed with
irrigation solutions and intracanal medicaments in in vitro
and in vivo conditions in order to evaluate the antibacte-
riological effects of the individual steps in the endodontic
procedure.
Conclusion
ProTaper instrumentation was more effective in reducing
bacterial numbers in infected root canals than stainless
steel hand files and the other tested Ni-Ti instruments.
Acknowledgements
This study was supported by the Hacettepe University
Scientific Research Fund, project number 07D01201001,
and presented at the 4th Scientific Symposium of the
Endodontic Societies of Turkey and Kosovo 23–26 April
2009, Antalya, Side, Turkey, as an oral presentation.
References
1. Kakehashi S, Stanley HP, Fitzgerald RJ. The effects of
surgical exposure of dental pulps in germ-free and con-
ventional laboratory rats. Oral Surg 1965; 20: 340–9.
2. Sundqvist G. Ecology of the root canal flora. J Endod
1992; 18: 427–30.
3. Gomes BPFA, Lilley JD, Drucker DB. Variations in the
susceptibilities ofcomponents of the endodontic micro-
flora of biomechanical procedures. Int Endod J 1996; 29:
235–41.
4. Haapasalo M, Endal U, Zandi H, Coil JM. Eradication of
endodontic infection by instrumentation and irrigation
solutions. Endod Topics 2005; 10: 77–102.
5. Siqueira JF Jr, Rocas IN, Favieri A, Lima KC. Chemome-
chanical reduction of the bacterial population in the root
canal after instrumentation and irrigation with 1%,
2.5%, and 5.25% sodium hypochlorite. J Endod 2000;
26: 331–4.
6. Siqueira JF Jr, Rocas IN, Santos SR, Lima KC, Magalhaes
FA, de Uzeda M. Efficacy of instrumentation techniques
and irrigation regimens in reducing the bacterial popula-
tion within root canals. J Endod 2002; 28: 181–4.
7. Shuping GB, Ørstavik D, Sigurdsson A, Trope M. Reduc-
tion of intracanal bacteria using nickel titanium rotary
instrumentation and various medications. J Endod 2000;
26: 751–5.
8. Byström A, Sundqvist G. Bacteriological evaluation of
the efficacy of mechanical root canal instrumentation in
endodontic therapy. Scand J Dent Res 1981; 89: 321–8.
9. Ørstavik D, Kerekes K, Molven O. Effects of extensive
apical reaming and calcium hydroxide dressing on bacte-
132
M. Gorduysus et al.
rial infection during treatment of apical periodontitis: a
pilot study. Int Endod J 1991; 24: 1–7.
10. Dalton BC, Ørstavik D, Philips C, Pettiette M, Trope M.
Bacterial reduction with nickel-titanium rotary instru-
mentation. J Endod 1998; 24: 763–7.
11. Baugh D, Wallace J. The role of apical instrumentation
in root canal treatment: a review of the literature.
J Endod 2005; 31: 333–40.
12. Love RM. Regional variation in the root dentinal tubule
infection by Streptococcus gordonii. J Endod 1996; 22:
290–3.
13. Matsuo T, Shirakami T, Ozaki K, Nakanishi T, Yumoto
H, Ebisu S. An immunohistological study of the localiza-
tion of bacteria invading root pulpal walls of teeth with
periapical lesions. J Endod 2003; 29: 194–200.
14. Aydın C, Tunca YM, Senses Z, Baysallar M, Kayaoglu G,
Ørstavik D. Bacterial reduction by extensive versus
conservative root canal instrumentation in vitro. Acta
Odontol Scand 2007; 65: 167–70.
15. Siqueira JF Jr, Lima KC, Magalhaes FA, Lopes HP, de
Uzeda M. Mechanical reduction of the bacterial popula-
tion in the root canal by three instrumentation tech-
niques. J Endod 1999; 25: 332–5.
16. Kerekes K, Tronstad L. Long-term results of endodontic
treatment performed with a standardized techinique.
J Endod 1979; 5: 83–90.
17. European Society of Endodontology. Consensus report
of the European Society of Endodontology on quality
guidelines for endodontic treatment. Int Endod J 1994;
27: 115–24.
18. Card SJ, Sigurdsson A, Orstavik D, Trope M. The effec-
tiveness of increased apical enlargement in reducing
intracanal bacteria. J Endod 2002; 28: 779–83.
19. Lam PP, Palamara JE, Messer HH. Fracture strength of
toot roots following canal preparation by hand and
rotary instrumentation. J Endod 2005; 31: 529–32.
20. Rollison S, Barnett F, Stevens RH. Efficacy of bacterial
removal from instrumented root canals in vitro
related to instrumentation technique and size. Oral
Surg Oral Med Oral Pathol Oral Radiol Endod 2002;
94: 366–71.
21. Deplazes P, Peters O, Barbakow F. Comparing apical
preparations of root canals shaped by nickel-titanium
hand instruments. J Endod 2001; 27: 196–202.
22. Ahlquist M, Henningsson O, Hultenby K, Ohlin J. The
effectiveness of manual and rotary techniques in the
cleaning of root canals: a scanning electron microscopy
study. Int Endod J 2001; 34: 533–7.
23. Schafer E, Lohmann D. Efficiency of rotary nickel
titanium FlexMaster instruments compared with
stainless steel hand K-Flexofile – Part 1. Shaping ability
in simulated curved canals. Int Endod J 2002; 35: 505–
13.
24. Dahlen G, Haapasalo M. Microbiology of apical peri-
odontitis. In: Orstavik D, Pitt Ford TR, eds. Essential
© 2010 The Authors
Australian Endodontic Journal © 2010 Australian Society of Endodontology
M. Gorduysus et al.
endodontology: prevention and treatment of apical
periodontitis. 1st ed. Oxford: Blackwell Sciences Ltd.;
1998. pp. 106–25.
25. Sjogren U, Figdor D, Spangberg L, Sundqvist G. The
antimicrobial effect of calcium hydroxide as a
© 2010 The Authors
Australian Endodontic Journal © 2010 Australian Society of Endodontology
Elimination of Bacteria from the Root Canal
short-term intracanal dressing. Int Endod J 1991; 24:
119–25.
26. Beer R, Baumann MA, Kim S. Endodontology-color
atlas of dental medicine. New York: Thieme Verlag;
2000.
133