Indian Journal of Dental Research

: 2015  |  Volume : 26  |  Issue : 1  |  Page : 67--71

Apical extrusion of Enterococcus faecalis using three different rotary instrumentation techniques: An in vitro study

Sonali Taneja, Manju Kumari, Madhumita Barua, Chetna Dudeja, Meeta Malik 
 Department of Conservative Dentistry and Endodontics, I.T.S Centre for Dental Studies and Research, Ghaziabad, Uttar Pradesh, India

Correspondence Address:
Sonali Taneja
Department of Conservative Dentistry and Endodontics, I.T.S Centre for Dental Studies and Research, Ghaziabad, Uttar Pradesh


Aims: To compare the apical extrusion of Enterococcus faecalis after instrumentation with three different Ni-Ti rotary instruments- An in vitro study. Settings and Design: In vitro study Methods and Material: Forty freshly extracted mandibular premolars were mounted in bacteria collection apparatus and root canals were contaminated with a suspension of Enterococcus faecalis. The contaminated teeth were divided into 4 groups of 10 teeth each according to rotary system used for instrumentation: Group1: Hyflex files, Group 2: GTX files, Group 3: Protaper files and Group 4: control group (no instrumentation). Bacteria extruded after preparations were collected into vials and microbiological samples were incubated in BHI broth for 24 hrs. The colony forming units were determined for each sample. Statistical Analysis Used: Statistical analysis was done using one way ANOVA followed by post hoc independent �DQ� t�DQ� test. Results: GTX files extruded least amount of bacteria followed by Hyflex files. Maximum extrusion of E. faecalis was seen in rotary Protaper group. Conclusion: Least amount of extrusion was seen with GTX files followed by Hyflex files and then rotary Protaper system.

How to cite this article:
Taneja S, Kumari M, Barua M, Dudeja C, Malik M. Apical extrusion of Enterococcus faecalis using three different rotary instrumentation techniques: An in vitro study.Indian J Dent Res 2015;26:67-71

How to cite this URL:
Taneja S, Kumari M, Barua M, Dudeja C, Malik M. Apical extrusion of Enterococcus faecalis using three different rotary instrumentation techniques: An in vitro study. Indian J Dent Res [serial online] 2015 [cited 2023 Sep 22 ];26:67-71
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The goal of endodontic treatment is complete debridement of root canal and to maintain healthy periapical tissues. During chemomechanical preparation, pulp tissue fragments, necrotic tissue, micro-organisms, and intracanal irrigants may extrude through the apical foramen into periradicular tissues and induce an inflammatory reaction. [1] Bacteria extruded mainly include Gram-positive, Gram-negative bacteria and obligate anaerobes. [2] Micro-organisms seen in root canal failure cases include Enterococcus faecalis, Propionibacterium alactolyticus, and Propionibacterium propionicum. [3] E. faecalis has been identified as a species most commonly recovered from post-treatment diseases. [4]

All preparation techniques and instruments have reported to extrude infected debris, even when preparation is maintained short of apical terminus. [5],[6],[7],[8],[9] Er et al. [10] and Kustarci et al. [11] reported that intracanal bacteria can be extruded apically along with debris during instrumentation.

In the last decade, rotary endodontics is gaining popularity. Different rotary systems vary in instrument design and thus differences exist in terms of apical extrusion of debris. Hyflex files (CM wire) and GTX (M wire technology) are among the new rotary systems with good cutting efficiency and increased resistance to cyclic fatigue. Rotary Protaper files with variable taper have been widely used till date. Various studies have reported that Protaper produces more extrusion of debris as compared to the profile. [12],[13] Kustarci et al. reported that Protaper extrudes more debris as compared to K 3 rotary instruments. [14] Till date, no study has been done comparing the apical extrusion of bacteria after instrumentation with Hyflex files and GTX files. Hence, the aim of this study was to compare the apical extrusion of E. faecalis using Hyflex files, GTX files, and rotary Protaper files.


Freshly extracted human permanent single-rooted mandibular premolar teeth were collected from patients above 50 years of age requiring extractions due to periodontal reasons. Teeth with canal curvature 0-10° and apical diameter confirming to 15 K file (Dentsply Maillefer, Ballaigues, Switzerland) were selected. For this, a size 15 K file was inserted in the canal until it was just visible but not extending beyond the apical foramen and showed tug back. Teeth were decoronated at 14 mm so that the working length was 13 mm. Two coats of nail varnish were applied to an external surface of the root, and a hole was created in the region of the apical foramen using 10 K file to maintain the apical patency.

Test apparatus

Teeth were forced through the rubber stopper of glass vials and remaining gap was sealed with self-cure acrylic resin. A 24 gauge needle was inserted into the rubber stopper to equalize air pressure [Figure 1]. Entire apparatus was sterilized in an autoclave. Before experiment, vials were filled with normal saline.

Preparation of Enterococcus faecalis {Figure 1}

One ml of pure culture of E. faecalis (ATCC 29212) grown in BHI broth (Himedia Laboratories Pvt. Ltd; Mumbai, India) for 24 h was added to fresh BHI broth. McFarland standard number 0.5 was used to evaluate the broth to ensure that number of bacteria were 1.5 × 10 8 CFU/ml. Root canals were filled with 10 μl of suspension and carried down the length of the canal using a 15 K file. Incubation was done at 37°C for 24 h. While the samples were infected with E. faecalis, simultaneous inoculation of the broth was also done on blood agar plate to see the viability of the strain in the broth which was used to infect the root canal. This culture plate showed growth of E. faecalis indicating that bacteria in the broth were alive and multiplying.


Samples were equally divided into 4 groups for instrumentation with different techniques:

Group 1: Hyflex CM (Coltene/Whaledent Inc., USA) 25/0.08 was used as orifice opener and moved slowly forward without pressure in pecking motion. Next, 20/0.04 file was used with pecking motion till WL followed by 25/0.04. Then, 20/0.06 file used to shape middle part of root canal followed by 30/0.04 and 30/0.06 till WLGroup 2: GTX (Dentsply, Tulsa Dental Specialities, Tulsa, OK) 20/0.06 was used, passed to WL in 1-3 passes followed by 30/0.06Group 3: Protaper (Dentsply Maillefer, Switzerland) SX was used as orifice shaper followed by S1 till coronal two-thirds of the canal. Next, S1, S2 were used to full WL Followed by the use of F1, F2, F3 till full WLGroup 4: No instrumentation (control).Instrumentation was done according to manufacturer's instructions. Scouting was done with 10 K file after each instrument to ensure canal patency. Master apical file and taper was kept constant in all the groups. A volume of 7 ml saline was used as irrigant during instrumentation of each sample. Entire procedure was performed under a class I laminar airflow cabinet to prevent airborne bacterial contamination. After instrumentation, 0.1 ml saline was taken from each vial and incubated in BHI agar (Himedia Laboratories Pvt. Ltd; Mumbai, India) at 37°C for 24 h. Bacterial colonies were counted using colony counter. Statistical analysis was done using SPSS software (Version 20, Economic & Statistical Division, MIcrosoft Corporation, Chicago). It was analyzed using One-way ANOVA and post hoc independent t-test. The level of statistical significance was set at P = 0.05. Results were expressed as CFU/ml.


Data regarding the number of bacteria extruded are presented in [Table 1] and Graph 1. The results indicated that all instrumentation groups caused measurable apical extrusion of bacteria. GTX files showed least apical extrusion of bacteria followed by Hyflex files [Figure 2] and [Figure 3]. Maximum bacterial extrusion was seen in rotary Protaper group and no extrusion was seen in control group as shown in [Figure 4] and [Figure 5] respectively. Pairwise comparison among different groups showed P < 0.05 indicating significant differences between the groups.{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Table 1} [INLINE:1]


The main objective of root canal therapy is to prevent and treat peri-radicular inflammation by eliminating micro-organisms from the root canal. [15] During biomechanical instrumentation, necrotic debris, bacteria may enter the periapical tissues and disrupt the balance between microbial aggression and host defenses leading to acute exacerbations and flare-ups. Thus, the aim of the present study was to assess the extrusion of intracanal E. faecalis as a result of canal shaping using different rotary instruments. The null hypothesis was that there is no difference in apical bacterial extrusion when biomechanical preparation was done using GTX, Hyflex, and Protaper rotary systems.

Methodology employed was similar to that used by Er et al. [10] E. faecalis was used as the bacteriological marker as it is implicated in persistent root canal infections and is identified as the species most commonly recovered from root canals of teeth post treatment disease. [15] It is non-fastidious, easy to grow, facultative aerobic bacterium and is reported to survive alone without symbiotic support from other bacteria. [15] Standardized tooth model was used to decrease the number of variables and to increase the probability that the amount of apically extruded debris was due to instrumentation alone. Instrumentation was performed by one trained operator. [15] Careful selection of teeth was done according to tooth type, canal size at the working length and canal curvature to ensure that number of extruded bacteria was due to instrumentation technique and not due to tooth morphology. [10] In addition, teeth were decoronated to keep root canals similar in length and to create easy reference point for working length. [9] Instrumentation was confined to 1 mm short of the apical foramen. Beeson et al. reported that when instrumentation was performed till apical foramen, more debris was forced apically than when instrumentation is 1 mm short of it. [16] Normal saline was used as irrigating solution as it has no antibacterial effect. Hence, extrusion and elimination of bacteria depended only on mechanical action of instruments. [15] As the bacteria can also be introduced into the periapical tissue during irrigating process, the passive injection of irrigant was done in order to reduce the bacteria forcing out of apical foramen with anything other than the preparation system being tested. [15]

In the present study, the amount of extrusion seen in GTX files and Hyflex files was significantly less than Protapers. Among GTX and Hyflex, GTX showed the least amount of extruded bacteria. Thus, the null hypothesis was rejected. This may be attributed to the presence of variable width lands, wider flutes in GTX files that create deeper chip space allowing space for coronal removal of bacteria. Blade angles are open to consistent 30° along the length doubling chip space between flutes. Land widths at tip and shank ends are half the sizes of land in the middle region, allowing rapid cutting without transportation. Variable width lands virtually eliminate taper lock in the canal. Chance of transporting a root canal with a rotary file that has non-cutting tip and radial lands is minimal. This might have been responsible for less apical extrusion of bacteria with GTX files as compared to Hyflex and rotary Protaper files.

Hyflex files machined from a wire (termed CM wire) have double fluting, symmetrical cross-section, variable pitch, non-cutting tip, negative rake angle. According to the manufacturer, cutting profile of these files facilitates penetration in canal and presents root canal shape corresponding with original anatomy. They provide precise apical finishing, leaving structural integrity of root canal intact after RCT. In the present study, Hyflex files showed more extrusion of bacteria as compared to GTX. This may be due to the absence of radial lands and the increased number of files used during instrumentation than GTX files.

The large amount of extruded bacteria in rotary Protaper system may be attributed to its long pitch, absence of radial lands and larger taper apical files. The taper of F3 is 0.09. Elmsallati et al. reported that the file with short pitch design extrudes less debris. [17] Protapers have shown to remove a substantial amount of dentin in a short period of time. [12],[13],[18],[19] Furthermore, shaping files, S1 and S2 that reach the full working length have partially active tips as compared to other two systems used in the present study. Moreover, Tasdemir et al. also reported largest amount of debris extrusion by Protaper system as compared to Mtwo and Race systems. [19] This was attributed to the faster, aggressive cutting with Protapers and long pitch design that causes a greater amount of bacteria to be extruded.

Results of the study cannot be correlated to any other study as no study has been performed till date comparing extrusion of bacteria by Hyflex, GTX, and Protaper files. Many factors affect the amount of extruded bacteria including instrument type, instrumentation technique, instrument size, preparation end point and irrigating solution. [10] Rotary instrumentation extrudes less bacteria than hand instrumentation as it tends to direct debris towards the orifice by packing the debris into the flutes of the instrument, avoiding its compaction in root canal. [1] Furthermore, in rotary instrumentation, file contacts apical area for a shorter period of time, and the rotational speed and torque is fixed. [18] Crown down technique extrudes less bacteria than step back technique owing to early flaring of the coronal part of the preparation that removes debris and improves instrument control in the apical third. [20] It must be emphasized that results of this study should not be directly extrapolated to clinical situations as periapical tissues may serve as a natural barrier, inhibiting debris extrusion. Results may also differ because of positive and negative pressure at the apex, and with normal and pathological periapical tissues. Furthermore, results of the present study cannot be applied to immature roots and open apices. [19] Under the limitations of the present study, all rotary systems used showed significant apical extrusion of bacteria. Least amount of extrusion was seen with GTX files followed by Hyflex files and then rotary Protaper system. Further in vivo research in this direction could provide more insight into biologic factors associated and focus on bacterial species that essentially play a major role in post instrumentation flare-ups.


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