Bacterial penetration and proliferation in root canal dentinal tubules after applying dentin adhesives in vitro

Endodontic treatment is aimed at eliminating infection and preventing bacterial regrowth in the root canal and dentinal tubules. In the present study the ability of two dentin adhesives to prevent bacterial penetration and subsequent proliferation in dentinal tubules was evaluated. Cylindrical root specimens prepared from freshly extracted bovine teeth were used in an in vitro model of dentinal tubule infection. After removal of the smear layer the intracanal dentinal tubules of the specimens were acid-etched and treated with either Gluma or EBS. Untreated specimens served as controls. Specimens were infected with Enterococcus faecalis and incubated in Brain Heart Infusion for 21 days. Powder dentin samples obtained from within the canal lumina, using ISO 025 to 033 burs, were examined for the presence of vital bacteria by inoculating on agar plates and counting colony-forming units. A significant difference was found between the experimental groups and the untreated group. After application with Gluma specimens showed the least viable bacteria in dentinal tubules. Data suggested that dentin adhesives reduced bacterial invasion into dentin and therefore have a potential role in endodontic treatment.     

A comparison of the antimicrobial efficacy of three calcium hydroxide formulations on human dentin infected with Enterococcus faecalis

This study compared the antibacterial efficacy of three different formulations of calcium hydroxide by using human dentin specimens that were infected with Enterococcus faecalis. After exposure to three forms of calcium hydroxide (calcium hydroxide mixed with distilled water, calcium hydroxide mixed with 0.2% chlorhexidine, and calcium hydroxide mixed with camphorated paramonochlorophenol) for 7 days, dentin powder from the infected specimens was obtained and assessed for bacterial quantity by spectrophotometry. It was found that calcium hydroxide mixed with camphorated paramonochlorophenol killed all of the Enterococcus faecalis inside the dentinal tubules. This result was better than that obtained with calcium hydroxide mixed with distilled water or with 0.2% chlorhexidine (p < 0.05). Calcium hydroxide mixed with distilled water and calcium hydroxide mixed with 0.2% chlorhexidine were ineffective against these bacteria.     

A new noninvasive model to study the effectiveness of dentin disinfection by using confocal laser scanning microscopy

Introduction

The present study was designed to develop a standardized model for quantification of the effectiveness of dentin disinfection by different antibacterial solutions including a new root canal irrigant, Qmix.

Methods

Dentinal tubules from the root canal side in semicylindrical dentin specimens were infected with Enterococcus faecalis by centrifugation of the bacterial suspension into the tubules. Scanning electron microscopy (SEM) was used to verify the presence of bacteria in dentin. The outer side of dentin pieces was closed, and the specimens were subjected to 1-minute and 3-minute exposure to sterile water, 1%, 2%, 6% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and Qmix. Confocal laser scanning microscopy (CLSM) and viability staining were used to quantitatively analyze the proportions of dead and live bacteria inside dentin.

Results

A heavy invasion by E. faecalis was detected by both SEM and CLSM throughout the dentinal tubules. The amount of dead cells in dentin increased with increasing NaOCl concentration and time of exposure (P < .05). Qmix was equally effective in killing bacteria in dentin as 6% NaOCl; more than 40% and 60% of the bacteria were killed by both at 1 minute and 3 minutes, respectively. One percent and 2% NaOCl and 2% CHX killed 20%–30% and 30%–40% bacteria after 1 and 3 minutes of exposure, respectively, with no statistically significant differences among the 3 agents (P > .05). In the control group, which was treated with sterile water, only 4%–6% of the bacteria were dead.

Conclusions

Centrifugation helped to create a heavy, evenly distributed infection deep into the dentinal tubules. The new model made it possible to compare the effectiveness of several disinfecting solutions in killing bacteria inside dentin by a noninvasive CLSM method.     

In vitro infection and disinfection of dentinal tubules

An in vitro model for dentinal tubule infection of root canals was developed. Cylindrical dentin specimens, 4 mm high with a diameter of 6 mm and a canal 2.3 mm wide, were prepared from freshly extracted bovine incisors. The cementum was removed from all dentin blocks. The tubules were opened by four-minute treatments with 17% EDTA and 5.25% NaOCl before being infected with Enterococcus faecalis ATCC 29212 in yeast extract-glucose broth. Bacteria rapidly invaded the tubules. After three weeks of incubation, a heavy infection was found 400 micron from the canal lumen, and the front of the infection reached 1000 micron in some blocks. Camphorated paramonochlorophenol (CMCP) and a calcium hydroxide compound, Calasept, were tested for their disinfecting efficacy toward E. faecalis-infected dentin. Liquid CMCP rapidly and completely disinfected the dentinal tubules, whereas CMCP in gaseous form disinfected tubules less rapidly. Calasept failed to eliminate, even superficially, E. faecalis in the tubules. The method used in bacteriological sampling allowed for sequential removal of 100-micron-thick zones of dentin from the central canal toward the periphery. Control specimens were uniformly infected and yielded growth in bur samples up to some 500 microns from the surface. The model proved quite sensitive and seems suitable for in vitro testing of root canal medicaments.     

Effectiveness of three endodontic irrigants at various tubular depths in human dentin

Bacteria from infected root canals can invade dentinal tubules, thus dentin disinfection is an important aspect of endodontic therapy. This study compares three endodontic irrigants for efficiency in killing bacteria established within human dentinal tubules. Root canals in extracted teeth were prepared and sterilized. Broth cultures of Enterococcus faecalis were allowed to grow within the canals to penetrate dentinal tubules. The infected canals were exposed individually to each of the irrigants for 1 min. Irrigants were 0.525% sodium hypochlorite, Tubulicid (0.2% EDTA), and 0.12% chlorhexidine (Peridex). Sterile water was the control. Viable bacteria were analyzed by drilling incrementally into dentin from the cementum toward the canal. Smaller diameter drills were used for each depth. Shavings were cultured at three depths, for each of three root levels: coronal, midroot, and apical. Although considerable variation occurred between roots, sodium hypochlorite seemed to be superior. Tubulicid and Peridex were better than water. More bacteria remained viable at greater distances from the pulp. These observations apparently apply to all levels in the canal.     

Antimicrobial activity of several calcium hydroxide preparations in root canal dentin.

The purpose of this study was to evaluate the antimicrobial activity of several calcium hydroxide (Ca(OH)2) preparations in root canal dentin infected with Enterococcus faecalis. Roots of extracted bovine incisors were prepared to standardized cylindrical test specimens of 5 mm in height; the smear layer was removed, and the specimens were incubated for 24 h at 37 degrees C in bacteriological culture medium that contained 7.0 x 10(4) colony forming units per milliliter of E. faecalis. The specimens were mounted in individual 4-mm diameter culture wells, and the test material was applied to fill the canal lumen. There were five treatment groups: group 1, a thick mixture of Ca(OH)2 USP (1.0 g/ml H2O); group 2, a thin mixture of Ca(OH)2 USP (0.1 g/ml H2O); group 3, Pulpdent TempCanal paste; group 4, sterile H2O (positive control); and group 5, 25 dentin specimens in sterile, uninoculated brain-heart infusion broth that were included as negative controls. Quantitative microbiological analysis of dentin at various depths was completed after 24 h. All groups showed a significant (p < 0.001) decrease in numbers of E. faecalis in all depths of dentin compared with the control. Groups 2 and 3 demonstrated significantly greater antimicrobial activity (73%-86% reduction) at all depths of dentin tested compared with group 1 (13%-26%) (p < 0.05). These results suggest that Ca(OH)2 can decrease the numbers of E. faecalis at all depths of dentinal tubules within 24 h and that thin preparations of Ca(OH)2 may be more effective in the elimination of E. faecalis from dentinal tubules than thick preparations.     

Antimicrobial activity of Ca(OH)2 containing pastes with Enterococcus faecalis in vitro

Sixty-eight standardized human root specimens were infected with Enterococcus faecalis for 3 wk after removal of the smear layer. After 3 wk of infection, the smear layer was reformed and in half of the specimens, the smear layer was again removed. Aqueous Ca(OH)2 paste and silicone oil based Ca(OH)2 paste were used as the test medications. The specimens were divided into four groups (i.e. (a) nonsmeared aqueous calcium hydroxide group, (b) nonsmeared silicon oil-based calcium hydroxide group, (c) smeared aqueous calcium hydroxide group, and (d) smeared silicon oil-based calcium hydroxide group. Medications were placed in the canals for 7 days. After removal of medications dentin chips were collected and incubated. The quantity of bacteria present was assessed. All calcium hydroxide pastes were effective in the elimination of bacteria in the dentinal tubules, except in the smeared group with silicone oil-based calcium hydroxide.     

Effect of electrophoretically activated calcium hydroxide on bacterial viability in dentinal tubules –in vitro

Abstract –  To evaluate the ability of electrophoretically activated calcium hydroxide (CH) to eliminate bacteria in dentinal tubules. In an in vitro model of dentinal tubule infection, 18 cylindrical root specimens prepared from freshly extracted bovine teeth were used. After removal of the smear layer, intracanal dentinal tubules were infected with Enterococcus faecalis for 21 days. In 12 specimens, CH paste was placed in the root canals for 7 days. In six of these, an electrophoretic current (10 mA per 10 min), using two electrodes, was applied after placing the medicament in the canal. Powder dentin samples obtained from within the canal lumina using ISO 025, 027, 029, 031 and 033 burs were examined for the presence of vital bacteria by inoculating agar plates and counting colony forming units. anova with repeated measures was used to analyze results. A significant difference was found between experimental groups and the positive control group. CH and electrophoretically activated CH significantly ( P  < 0.001) reduced bacterial viabilities in dentinal tubules to a depth of 200  μ m. Treatment with electrophoresis was significantly ( P  < 0.001) more effective than pure CH in depths of 200–500  μ m. Specimens treated with electrophoretically activated CH showed no viable bacteria in dentinal tubules to a depth of 500  μ m from the root canal space within 7 days. The time required for treatment of pulpal infection root resorption may be decreased, thus minimizing the risk of coronal fractures in young patients with traumatized teeth.     

Dentinal tubule disinfection using three calcium hydroxide formulations

The objective of this study was to determine the antibacterial efficacy of three calcium hydroxide (CH) formulations using an in vitro model of Enterococcus faecalis dentinal tubule infection. CH mixed with water (CH), CH mixed with iodine-potassium iodide (CH+IKI), and CH mixed with iodoform and silicone oil (Metapex) were tested. Human cylindrical dentin specimens infected with E. faecalis were filled with disinfectants and incubated for 1 week. Dentin powder samples collected with ISO 018 burs showed a statistically significant reduction in E. faecalis for all three experimental groups in comparison with untreated control specimens. Statistically significant differences were also found between the three experimental groups. Metapex was the most effective dentinal tubule disinfectant, followed by CH+IKI and then CH. Similar results were observed at greater dentin tubule depths (ISO 021 burs) with the exception that intracanal treatment with CH resulted in significantly higher numbers of E. faecalis in comparison with untreated control specimens.     

Efficacy of calcium hydroxide: chlorhexidine paste as an intracanal medication in bovine dentin

The purpose of this study was to evaluate the antibacterial efficacy of an intracanal medication composed of calcium hydroxide with 2% chlorhexidine. Dentin from 24 bovine incisors was used. The incisors were made into standardized cylindrical segments of dentin and infected with Enterococcus faecalis. They were then treated with an intracanal paste composed of calcium hydroxide and sterile water or an intracanal paste composed of calcium hydroxide and 2% chlorhexidine for 1 week. Dentin shavings were collected, suspended in solution, and spread on brain-heart infusion agar. After incubation, colony-forming units were enumerated. The amount of bacteria per mg of dentin was determined. The calcium hydroxide paste with 2% chlorhexidine was significantly more effective at killing E. faecalis in the dentinal tubules than calcium hydroxide with water.     

A laboratory study of the effect of calcium hydroxide mixed with iodine or electrophoretically activated copper on bacterial viability in dentinal tubules

AIM: The aim of this laboratory study was to evaluate the ability of calcium hydroxide (CH), CH/iodine-potassium iodide (IKI) and electrophoretically activated copper to kill bacteria in dentinal tubules. METHODOLOGY: In an in vitro model of dentinal tubule infection, 42 cylindrical root specimens, prepared from freshly extracted bovine teeth were used. After removal of the smear layer, intracanal dentinal tubules were infected with Enterococcus faecalis for 3 weeks. CH alone or preparations of CH with copper or IKI were placed in the root canal for 1 week. In specimens containing copper/CH, an electrophoretic current(5 mA/5 min) was applied using two electrodes follow-ing placement of the medicament in the canal. Powder dentine samples obtained from the canal wall using ISO sizes: 025, 027, 029, 031 and 033 burs were examined for the presence of viable bacteria by inoculating agar plates and counting colony forming units (cfu). RESULTS: A significant difference was found between the experimental groups and the positive control group. CH and CH/IKI significantly (P < 0.001)reduced bacterial viability in dentinal tubules to a depth of 200 microm. Specimens with CH/IKI had significantly fewer viable bacteria than CH alone in tubules between the depths of 200-500 microm. Treatment with CH/copper and electrophoresis was most effective: specimens showed no viable bacteria in dentinal tubules to a depth of 500 microm from the root-canal space. CONCLUSIONS: IKI or electrophoretically activated copper additives can significantly improve the antibacterial properties of CH in dentinal tubules.     

Antimicrobial substantivity of chlorhexidine-treated bovine root dentin

Previous studies have demonstrated antimicrobial substantivity in root canal dentin up to 7 days after treatment with chlorhexidine. This in vitro study assessed the antimicrobial substantivity of chlorhexidine-treated bovine root dentin over a period of 21 days. Sixty standardized bovine root sections were randomly divided into three equal groups, and their canals immersed in one of the following solutions: (i) sterile saline; (ii) 2.5% NaOCl; or (iii) 0.2% chlorhexidine (CHX). Half the specimens in each group were treated with the solution for 5 min and the other half for 7 days. After solutions were removed, the specimens were incubated at 37 degrees C in Brain Heart Infusion broth containing Enterococcus faecalis (ATCC 29212). A fresh inoculum was added to the broth every other day over a 21-day period. The canals were then enlarged with sterile burs, and the dentin shavings collected and cultured for the presence of cultivable bacteria in the dentinal tubules. Specimens treated with CHX for 7 days demonstrated significantly less dentin colonization by E. faecalis than the other specimens. CHX has potential as an intracanal medicament, if it can be applied for a period of at least 7 days.     

Dentin Extends the Antibacterial Effect of Endodontic Sealers against Enterococcus faecalis Biofilms

Introduction

The purpose of this study was to evaluate the antimicrobial effects of root canal sealers on Enterococcus faecalis biofilms in dentinal tubules by using a novel dentin infection model.

Methods

Cells of E. faecalis were introduced into the dentinal tubules by centrifugation and incubated in brain-heart infusion broth for 3 weeks. An equal thickness of AH Plus, Endosequence BC sealer (BC sealer), and pulp canal sealer EWT (PCEWT) was placed on the root canal wall of the dentin specimens for 1, 7, and 30 days in humid conditions at 37°C. Gutta-percha and water were used in a similar manner as the tested sealers. The proportions of dead and live bacteria inside the dentinal tubules after exposure to root canal sealers were assessed by confocal laser scanning microscopy.

Results

Significantly more bacteria were killed in the 3 sealer groups than in the 2 control groups (P < .05). BC sealer and AH Plus resulted in significantly more dead cells than PCEWT did. There was no statistically significant difference between BC sealer and AH Plus at any time point (P > .05). Thirty days of exposure to BC sealer and AH Plus resulted in significantly more dead bacteria in dentin than 7-day and 1-day exposures in the biofilms, whereas no statistically significant increase of the proportion of dead bacteria was detected between 7-day and 30-day PCEWT (P > .05).

Conclusions

The 3 endodontic root canal sealers had antibacterial effects against E. faecalis in the dentinal tubules. BC sealer and AH Plus had superior antibacterial effects compared with PCEWT. The antibacterial effects of sealers in dentinal tubules continued after setting.     

Antibacterial efficacy of calcium hydroxide, iodine potassium iodide, betadine, and betadine scrub with and without surfactant against E faecalis in vitro

OBJECTIVE: This study investigated the ability of endodontic irrigants and medicaments to eliminate Enterococcus faecalis from infected dentinal tubules, and whether their antimicrobial action was enhanced by surfactant. STUDY DESIGN: For the study, 5-mm dentin disks were sectioned from bovine incisor roots and infected with E faecalis. Lumens were instrumented, and 1 of 7 medicaments (10% Ca(OH) 2, Betadine, or IKI, each with or without surfactant, or Betadine Scrub) was used to flush and fill each lumen. Positive controls received saline. Specimens were incubated for 15 minutes or 24 hours. Quantitative microbiology of the remaining bacteria was performed and groups were compared using a 1-way ANOVA. RESULTS: The addition of surfactant did not enhance the antibacterial action of any medicament. When used as a 24-hour medicament, Ca(OH) 2 consistently failed to eliminate E faecalis, whereas both Betadine Scrub and IKI rendered 90% of samples sterile. IKI was the only agent shown to consistently eliminate E faecalis in a 15-minute time frame. CONCLUSION: Under the in vitro conditions of this study, IKI was able to eliminate E faecalis from bovine root dentin when used with a 15-minute contact time.     

Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules

The effect of endodontic irrigants and dressings was tested on bacteria in bovine dentin specimens experimentally infected with Enterococcus faecalis, Streptococcus sanguis, Escherichia coli, or Pseudomonas aeruginosa. Standardized, cylindrical dentin test pieces were prepared and cleaned by ultrasonic treatment with EDTA and sodium hypochlorite. The specimens were infected with the test organism for periods up to 14 days, and the degree of infection into the tubules was monitored using Brown & Brenn stain, scanning electron microscopy, and culturing of dentin dust from sequential bur samples starting from the pulpal side. E. faecalis rapidly infected the whole length of the tubules; S. sanguis required up to 2 weeks for complete infection; E. coli only penetrated to some 600 microns, even after prolonged incubation periods. P. aeruginosa infected dentin quickly, but apparently in very low numbers. E. faecalis persisted for at least 10 d after withdrawal of nutrient support, whereas the other 3 organisms died within 4 to 48 h. Endodontic medicaments were applied to infected specimen for comparison of antibacterial potency. Camphorated p-monochlorophenol was generally more efficient than Calasept, and of the irrigants tested, iodine potassium iodide appeared more potent than sodium hypochlorite or chlorhexidine. The presence of a smear layer delayed, but did not eliminate, the effect of the medicaments.     

In vitro yeast infection of human dentin

An in vitro model was developed for investigation of Candida albicans penetration into human dentinal tubules. The model consisted of a dentin disc mounted between two cuvettes that each had a circular opening facing the disc. The cuvettes were filled with Tryptic-Soy-Broth, and the pulpal side cuvette was inoculated with C. albicans and incubated at 37 degrees C in air until growth occurred in the uninoculated cuvette or up to 30 days. The system was also used with Enterococcus faecalis. Completely glue-covered dentin specimens served as negative controls. Brown & Brenn-stained histological preparations of the specimens were examined with light microscopy. The time needed before growth occurred in the uninoculated cuvette showed great variation with C. albicans, whereas E. faecalis penetrated within 1 to 5 days of incubation. Slight penetration both by hyphae and yeast cells was observed in specimens inoculated with C. albicans, whereas specimens inoculated with E. faecalis showed deep and effective penetration. This study demonstrates the penetration of dentin as a possible pathway of infection by C. albicans. However, dentin penetration by C. albicans was slow and limited in comparison with E. faecalis.     

In vitro evaluation of the antibacterial effect of four root canal sealers on dental biofilms

Objectives

To dynamically evaluate the effect of four root canal sealers on the killing of biofilms within dentinal tubules.

Materials and methods

Dentin blocks were prepared for infection of the dentinal tubules. Enterococcus faecalis VP3-181 and multi-species bacteria from two donors were cultured. After 3 days of incubation, the infected dentin specimens were rinsed with sterile water for 1 min and subjected to treatment. Additionally, multi-species bacteria from donor 1 were incubated for 3 weeks to allow biofilm maturation and then the specimens were subjected to treatment. Gutta-percha-treated dentin specimens comprised the control group. A root canal sealer (bioceramic sealers: EndoSequence BC Sealer, ProRoot Endo Sealer, or GuttaFlow Bioseal; and a traditional silicone-based sealer: Guttaflow 2) was spread onto the canal walls of the dentin. The specimens were examined with confocal laser scanning microscopy at 7, 30, or 60 days.

Results

In the 3-day-old biofilm group, the proportion of killed bacteria decreased significantly from the first 7 days of treatment to 60 days of treatment for all sealers (p?<?0.05). In the 3-week-old biofilm group, 60 days of exposure to bioceramic sealers resulted in more significant dead bacteria than 7-day exposures of the biofilms (p?<?0.05). Bioceramic sealers were more effective in killing bacteria than the GuttaFlow 2 sealer (p?<?0.05).

Conclusions

Calcium silicate–based sealers showed good antimicrobial effects against biofilms within dentinal tubules, especially in the first week in young biofilms. There is no substantive antibacterial activity observed for the examined root canal sealers against young dentinal tubule biofilms.

Clinical relevance

The bioceramic root canal sealers examined demonstrate minimal additional antibacterial effects after long-term exposure to young biofilms.

     

Effect of Smear Layer against Disinfection Protocols on Enterococcus faecalis–infected Dentin

IntroductionThis study examined the effect of the smear layer on the antibacterial effect of different disinfecting solutions in infected dentinal tubules.MethodsCells of Enterococcus faecalis were forced into dentinal tubules according to a previously established protocol. After a 3-week incubation period of infected dentin blocks, a uniform smear layer was produced. Forty infected dentin specimens were prepared and subjected to 3 and 10 minutes of exposure to disinfecting solutions including sterile water, 2% and 6% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), 17% EDTA, and QMiX (Dentsply Tulsa Dental, Tulsa, OK). The following combinations were also included: 2% NaOCl + 2% CHX, 2% NaOCl + QMiX, 6% NaOCl + QMiX, and 6% NaOCl + 17% EDTA + 2% CHX. Four other dentin specimens similarly infected but with no smear layer were subjected to 3 minutes of exposure to 2% CHX and 6% NaOCl for comparison. Confocal laser scanning microscopy and viability staining were used to analyze the proportions of dead and live bacteria inside the dentin.ResultsIn the presence of a smear layer, 10 minutes of exposure to QMiX, 2% NaOCl + QMiX, 6% NaOCl + QMiX, and 6% NaOCl + 17% EDTA + 2% CHX resulted in significantly more dead bacteria than 3 minutes of exposure to these same disinfecting solutions (P < .05). No statistically significant difference between 3 and 10 minutes was found in other groups (P > .05); 6% NaOCl + QMiX and 6% NaOCl + 17% EDTA + 2% CHX showed the strongest antibacterial effect. In the absence of a smear layer, 2% CHX and 6% NaOCl killed significantly more bacteria than they did in the presence of a smear layer (P < .05).ConclusionsThe smear layer reduces the effectiveness of disinfecting agents against E. faecalis in infected dentin. Solutions containing 6% NaOCl and/or QMiX showed the highest antibacterial activity.     

Molecular- and culture-based comparison of the effects of antimicrobial agents on bacterial survival in infected dentinal tubules

The aim of this study was to evaluate the effect of root canal obturation with or without prior calcium hydroxide (Ca(OH)(2)) or 2% chlorhexidine (CHX) on the persistence of bacteria or its DNA in infected dentinal tubules. Canals of 85 extracted teeth were instrumented and inoculated with 10(4) cells/mL of Enterococcus faecalis. Teeth were incubated at 37 degrees C for 21 days and divided into 3 groups of 25 teeth plus controls. Teeth in group 1 were obturated immediately with gutta-percha (GP) and AH-Plus (Maillefer, Dentsply, Tulsa, OK). In group 2, Ca(OH)(2) was placed for 7 days before obturation. In group 3, 10 minutes of irrigation was performed with CHX performed before obturation. After incubation, GP was removed, and dentin specimens were collected and analyzed with culturing and polymerase chain reaction (PCR). No growth occurred in any cultures. By using PCR, E faecalis was detected in fewer roots in group 3 than in groups 1 or 2 (chi(2), p = 0.05); 2% CHX treatment followed by obturation was more effective in removing E faecalis DNA than placement of Ca(OH)(2) or immediate obturation.     

Survival of Enterococcus faecalis in infected dentinal tubules after root canal filling with different root canal sealers in vitro

AIM: To investigate the ability of different endodontic sealers and calcium hydroxide to kill bacteria in experimentally infected dentinal tubules. METHODOLOGY: Fifty-six human root segments were enlarged to size 2 (ISO size 090) Largo Peeso Reamer. After treatment with 17% EDTA and 5% NaOCl for 4 min each, the specimens were infected with Enterococcus faecalis for 3 weeks. The roots were divided into eight groups and filled with gutta-percha and AH Plus (AH); Grossman's sealer (GS); Ketac-Endo (KE); Apexit (AP); RoekoSeal Automix (RSA); or RoekoSeal Automix with an experimental primer (RP), or calcium hydroxide (CH) only. One group of specimens was left unfilled for control (CT). Following storage in humid conditions at 37 degrees C for 7 days, the root canals were re-established with new sterile Largo size 2. Dentine samples from each canal were then collected using a sterile size 5 (ISO size 150) Largo Peeso Reamer. The number of colony-forming units (CFU) was determined for each sample. RESULTS: The mean log10 CFU in all test groups was significantly lower (P < 0.05) than that in the CT group. Root filling with AH and GS killed bacteria (mean CFU = 0) in the dentinal tubules. The mean log10 CFU for the CH group (0.53) was lower than that of RSA, AP, RP and KE (1.36, 1.40, 1.46 and 1.94, respectively), but only the difference between the CH and the KE groups was statistically significant (P < 0.05). CONCLUSION: Root fillings in vitro with gutta-percha and AH or GS were effective in killing E. faecalis in dentinal tubules. Other endodontic sealers, as well as CH, were less effective.