Preliminary evaluation of bioactive glass S53P4 as an endodontic medication in vitro

Calcium hydroxide is the "gold standard" endodontic medication, but it may fail to eliminate certain facultative bacteria and yeasts found in root canal systems. In this study, standardized bovine dentin blocks infected with Enterococcus faecalis were treated with an aqueous calcium hydroxide or bioactive glass S53P4 (BAG) powder suspension. While calcium hydroxide was ineffective, the BAG suspension eliminated the infection in the sampled dentin layers after 5 days. In a direct exposure test, preincubation with human dentin boosted the BAG-killing efficacy against E. faecalis ATCC29212, Candida albicans CCUG19915, Pseudomonas aeruginosa ATCC9027, Streptococcus sanguis ATCC10556, and S. mutans ATCC25175. BAG placed in the root canals of extracted teeth did not alter root-dentin pH. Consequently, the conditions under which the microbiota were killed were not pH-mediated. Further studies are ongoing to help clarify the antimicrobial BAG effect in the presence of dentin.     

Susceptibilties of two Enterococcus faecalis phenotypes to root canal medications

This study aimed to investigate and compare the efficacy of selected root canal irrigants and a medicament on a clinical isolate of Enterococcus faecalis grown as biofilm or planktonic suspension phenotype. A cell-dense pellet "presentation" prepared from planktonic phenotype was also tested. Each bacterial presentation was exposed to calcium hydroxide (pH 12.3), 0.2% chlorhexidine gluconate, 17% ethylene-diamine-tetra-acetic acid, 10% povidone iodine, or 3.0% sodium hypochlorite (NaOCl) for a range of time periods (1, 2, 4, 8, 15, 30, and 60 min). Phosphate buffered saline was used as a control agent. The difference in gradients of bacterial killing among the biofilm, planktonic suspension or pellet presentation was significant (p < 0.05) and dependent upon the test agent except in the case of NaOCl and calcium hydroxide where no difference could be detected. NaOCl was the most effective agent and achieved 100% kills for all presentations of E. faecalis after a 2 min contact time.     

Control of microorganisms in vitro by endodontic irrigants

The aim of this study was to determine the minimum inhibitory concentration (MIC) and antimicrobial effectiveness by the direct exposure test of 4 endodontic irrigants [1% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), 1% calcium hydroxide (Ca(OH)2; prepared with 1 g of Ca(OH)2 and 100 mL of sterile distilled water), a solution of Ca(OH)2 + detergent (HCT20)] for S. aureus, E. faecalis, P. aeruginosa, B. subtilis, C. albicans and a mixed culture. Microbial growth was analyzed by two methods: turbidity of the culture medium that was confirmed by Gram stain and subculture in a specific nutrient broth. In the dilution test, NaOCl solution showed MIC equal to 0.1% for S. aureus, E. faecalis, P. aeruginosa and C. albicans and equal to 1% for B. subtilis and the mixed culture. CHX (2%) presented MIC equal to 0.000002% for S. aureus, 0.02% for E. faecalis, B. subtilis, C. albicans and the mixed culture and 0.002% for P. aeruginosa. Ca(OH)2 solution (1%) showed MIC greater than 1% for all the microorganisms except P. aeruginosa for which it was equal to 1%. Calcium hydroxide solution + detergent showed MIC equal to 4.5 mL for S. aureus, P. aeruginosa, B. subtilis, C. albicans and the mixed culture and greater than 4.5 mL for E. faecalis. In the direct exposure test, NaOCl had better antimicrobial effectiveness for all microorganisms at all times. CHX (2%) was effective for S. aureus, E. faecalis and C. albicans at all times, and ineffective for P. aeruginosa, B. subtilis and the mixed culture. The other solutions showed the worst results.     

Susceptibility of oral Candida species to calcium hydroxide in vitro.

AIM: The susceptibility of common oral Candida species to saturated aqueous calcium hydroxide solution was studied. METHODOLOGY: The yeast species tested were C. albicans (16 strains). C. glabrata (three strains), C. guilliermondii (three strains), C. krusei (two strains), and C. tropicalis (two strains). At least one reference strain of each species was used; the others were clinical isolates either from persistent apical periodontitis or from marginal periodontitis. The susceptibility of Enterococcus faecalis (ATCC 29212) was studied for comparative purposes. Standardized inocula of the strains were incubated in aqueous calcium hydroxide solution, pH 12.4, for time-periods ranging from 5 min to 6 h. Volumes of 0.1 mL of the test suspension were cultured directly on Brucella blood agar and incubated in air at 37C. The plates were inspected for growth at 24 and 48 h and the colonies were counted. The time required to reduce the number of colony-forming units to less than 0.1% of the initial number was determined for each strain. RESULTS: The sensitivity of the C. albicans strains was generally low, with 16 h of incubation required to kill 99.9% of the colony-forming units. No differences in susceptibility between C. albicans strains isolated from root-canal infections and from periodontitis were found. Both strains of C. tropicalis were killed between 3 and 6 h of incubation, whilst strains of C. guilliermondii were killed after only 1020 min of incubation. All strains of C. glabrata survived 20 min, but not 1 h, of incubation, whilst 13 h were required to kill C. krusei. Compared with E. faecalis, all Candida spp. showed either equally high or higher resistance to aqueous calcium hydroxide. CONCLUSIONS: This study indicates that Candida spp. are resistant to calcium hydroxide in vitro, which may explain the isolation of yeasts from cases of persistent apical periodontitis.     

Elimination of Candida albicans infection of the radicular dentin by intracanal medications

Fungi have been associated with cases of secondary or persistent root canal infections. The purpose of this study was to evaluate the effectiveness of four intracanal medications in disinfecting the root dentin of bovine teeth experimentally infected with Candida albicans. Infected dentin cylinders were exposed to four different medications: calcium hydroxide/glycerin; calcium hydroxide/0.12% chlorhexidine digluconate; calcium hydroxide/camphorated paramonochlorophenol/glycerin; and 0.12% chlorhexidine digluconate/zinc oxide. Specimens were in contact with the medications for 1 h, 2 days, and 7 days. The viability of C. albicans after exposure was evaluated by specimen incubation in culture medium to compare the effectiveness of the medications in disinfecting dentin. Results showed that the specimens treated with calcium hydroxide/camphorated paramonochlorophenol/ glycerin paste or with chlorhexidine/zinc oxide paste were completely disinfected after 1 h of exposure. Calcium hydroxide/glycerin paste only consistently eliminated C. albicans infection after 7 days of exposure. Calcium hydroxide mixed with chlorhexidine was ineffective in disinfecting dentin even after 1 week of medication exposure. Among the medications tested, the calcium hydroxide/camphorated paramonochlorophenol/glycerin paste and chlorhexidine digluconate mixed with zinc oxide were the most effective in eliminating C. albicans cells from dentinal specimens.     

Effects of three different infant dentifrices on biofilms and oral microorganisms

The purpose of this work was to evaluate the effects of infant dentifrices: A--with lactoperoxidase, glucose oxidase and lactoferrin; B--with 1100 ppm of NaF and sodium lauryl sulfate; C--with extract of calendula. The dentifrices were test on biofilms formed in vitro from saliva and dental plaque of infants, using reference strains A. viscosus (ATCC 43146); C. albicans (ATCC 51501); L. casei (ATCC 4646); S. mitis (ATCC 49456); S. mutans (ATCC 25175); S. oralis (ATCC 35037); S. sanguis (ATCC 10586); S. sobrinus (ATCC 27609) and isolated clinically microorganisms C. albicans, S. mitis, S. mutans, S. oralis, S. sanguis, S. sobrinus and Lactobacillus sp. Twenty infants were chosen, who were beginning treatment at the Infants Clinic of the Pediatric Dentistry Department, Federal University of Rio de Janeiro. A pool of unstimulated saliva and a pool of dental plaque were collected from which biofilms were produced. Supernatants from each dentifrice were prepared and concentrated and diluted solutions of the dentifrices and a control sterile diluent were tested against the biofilms produced, for 1 and 3 minutes, and against the microorganisms. The results were statistically analyzed by the ANOVA and Tukey Test. After the exposure of the biofilms produced both from saliva and from dental plaque, to the dentifrice B concentrated and 1/2, for 1 and 3 minutes, the viable microorganisms count (CFU/ml), compared to the controls, was significantly reduced (p < 0.05). However, exposure to the dentifrices A and C concentrated and dentifrice B 1/4 and 1/8, for 1 and 3 minutes, was not significantly lethal to the biofilms. The dentifrices A and C, either concentrated or diluted (1/2 to 1/128) and the dentifrice B in the dilutions 1/16 to 1/128 did not have an antimicrobial effect on any microorganism evaluated. For all the microorganisms evaluated, the dentifrice B concentrated and in the 1/2 dilution showed a significant antimicrobial effect, when compared with the control (p < 0.05).     

Two methods to evaluate the antimicrobial action of calcium hydroxide paste.

The objective of this study was to analyze two methods for determining the antimicrobial effectiveness of (i) calcium hydroxide plus saline, (ii) calcium hydroxide plus polyethylene glycol, and (iii) calcium hydroxide plus camphorated paramonochlorophenol. Four microorganisms (Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (ATCC 6633)), one yeast (Candida albicans (ICB/USP-562)), and one mixture of these organisms were used. The strains were inoculated in Brain Heart Infusion (BHI) and incubated at 37 degrees C for 24 h. Two methods, the direct exposure test and the agar diffusion test were used to evaluate antimicrobial effects. For the direct exposure test (DET) 288 paper points were contaminated with the standard microbial suspensions and exposed to the intracanal dressings for 1, 24, 48, and 72 h. The points were immersed in Letheen Broth, followed by incubation at 37 degrees C for 48 h. An inoculum of 0.1 ml obtained from Letheen Broth was then transferred to 7 ml of BHI under identical incubation conditions, and microbial growth was evaluated. Pastes showed activity between 1 and 72 h, depending on the microorganism/mixture tested. For the agar diffusion test 36 Petri plates with 20 ml of BHI agar were inoculated with 0.1 ml of the same microbial suspension used for the DET, using sterile swabs that were spread on the medium. Five cavities were made in each of two agar plates (total = 10) and completely filled with one of the calcium hydroxide pastes. The plates were preincubated for 1 h at environmental temperature and then incubated at 37 degrees C for 24 to 48 h. The inhibition zone around each well was recorded in millimeters, and the results were submitted to ANOVA and Tukey test (alpha = 0.05). All intracanal dressings induced inhibition zones (range 5.0-10.0 mm). Data obtained showed that both the DET and agar diffusion test are useful in establishing the calcium hydroxide antimicrobial spectrum, thus improving infection control protocols. The direct exposure method is independent of other variables and is a practical laboratory procedure. A complete antimicrobial effect was observed after 48 h on indicator microorganisms, in both tests, irrespective of the calcium hydroxide paste vehicle.     

Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis

AIM: To determine the resistance of microorganisms associated with refractory endodontic infections to sodium hypochlorite used as a root canal irrigant. METHODOLOGY: Two strains each of Actinomyces naeslundii, Candida albicans and Enterococcus faecalis were tested as late logarithmic phase inocula, against sodium hypochlorite adjusted to 0.5, 1.0, 2.5 and 5.25% w/v. Contact times used were 0, 10, 20, 30, 60 and 120 s. In the case of E. faecalis, additional experiments used contact times of 1.0, 2.0, 5.0, 10.0 and 30.0 min. Anti-microbial action was halted by sodium thiosulphate addition. Survivors were measured primarily using viable counts on drop plates. Additionally, pour plates were used to count low colony-forming units (cfu) and dilutions to 10(-6) were used to count high cfu. RESULTS: All concentrations of NaOCl lowered cfu below the limit of detection after 10 s in the case of A. naeslundii and C. albicans. However, E. faecalis proved to be more resistant to NaOCl. Using 0.5% NaOCl for 30 min reduced cfu to zero for both strains tested. This compares with 10 min for 1.0%, 5 min for 2.5% and 2 min for 5.25% (P < 0.001). Regression analysis for the dependent variable log(e)(count + 1) with log(e)(time + 1) and concentration as explanatory variables gave rise to a significant interaction between time and concentration (P < 0.001). CONCLUSION: The published association of E. faecalis with refractory endodontic infection may result, at least partially, from high resistance of this species to NaOCl. This does not appear to be the case with A. naeslundii or C. albicans.     

Biofilm formation in medicated root canals

The hypothesis that Enterococcus faecalis resists common intracanal medications by forming biofilms was tested. E. faecalis colonization of 46 extracted, medicated roots was observed with scanning electron microscopy (SEM) and scanning confocal laser microscopy. SEM detected colonization of root canals medicated with calcium hydroxide points and the positive control within 2 days. SEM detected biofilms in canals medicated with calcium hydroxide paste in an average of 77 days. Scanning confocal laser microscopy analysis of two calcium hydroxide paste medicated roots showed viable colonies forming in a root canal infected for 86 days, whereas in a canal infected for 160 days, a mushroom-shape typical of a biofilm was observed. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed no differences between the protein profiles of bacteria in free-floating (planktonic) and inoculum cultures. Analysis of biofilm bacteria was inconclusive. These observations support potential E. faecalis biofilm formation in vivo in medicated root canals.     

Fungi in endodontic infections

Fungi are chemoorganotroph eukaryotic microorganisms that can take part in endodontic infections and thereby may participate in the etiology of periradicular diseases. They possess virulence attributes--including adaptability to a variety of environmental conditions, adhesion to a variety of surfaces, the production of hydrolytic enzymes, morphologic transition, biofilm formation, and evasion and immunomodulation of the host defense--that may play a role in the pathogenesis of periradicular diseases. Fungi have occasionally been found in primary root canal infections, but they seem to occur more often in the root canals of obturated teeth in which treatment has failed. Candida albicans is by far the fungal species most commonly isolated from infected root canals, and this species has been considered a dentinophilic microorganism because of its invasive affinity to dentin. C albicans has also been discovered to be resistant to some intracanal medicaments, such as calcium hydroxide. Its ability to invade dentinal tubules and resistance to commonly used intracanal medicaments may help to explain why C albicans has been associated with cases of persistent root canal infections. Some medicaments, such as chlorhexidine digluconate, calcium hydroxide combinations (with camphorated paramonochlorophenol or chlorhexidine), and EDTA, have the potential to be used as effective intracanal medications for patients in whom fungal infection is suspected.     

Effect of tissue fluids on hydrophobicity and adherence of Enterococcus faecalis to dentin

This in vitro study was carried out to determine (1) the hydrophobicity of selected oral bacteria, (2) the influence of growth media (saliva and serum) and mode of growth (planktonic or biofilm) on the hydrophobicity of Enterococcus faecalis, and (3) the influence of growth media and conditioning fluids on the adherence of E. faecalis to dentin. The ability to bind to a hydrocarbon phase (xylene) was used as an index of relative hydrophobicity of cells. Fluorescent microscopy-based technique was used to assay the bacterial adherence to dentin. Results showed that bacteria involved in the primary stage of oral biofilm formation such as Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis are relatively more hydrophobic than E. faecalis. The hydrophobicity of E. faecalis was significantly increased during starvation and biofilm mode of growth (p < .05). The adherence of E. faecalis to dentin was appreciably increased after starvation and when dentin was conditioned with saliva. It was observed that surface conditioning of dentin with saliva and starvation can enhance the adherence of E. faecalis to dentin. The findings from this study indicated that the coronal leakage of saliva and the physiologic state of microbes might play an important role in the adherence and biofilm formation of bacteria to root canal dentin.     

Effects of solutions used in infants' oral hygiene on biofilms and oral microorganisms

The purpose of this work was to evaluate the effects of oral hygiene solutions used for infants on biofilms formed in vitro from infants' saliva and dental plaque: ATCC reference strains A. viscosus; C. albicans; L. casei; S. mitis; S. mutans; S. oralis; S. sanguis; S. sobrinus and clinically isolated microorganisms (saliva) C. albicans, S. mitis, S. mutans, S. oralis, S. sanguis and S. sobrinus. After exposure of the oral biofilms to H2O2 diluted 1/4 to 1/16; and NaF 0.02 percent, concentrated and diluted 1/2, for 1 and 3 minutes, the viable count of microorganisms, compared to the controls was significantly reduced (p < 0.05). They also showed a significant antimicrobial effect for all the microorganisms evaluated, when compared to the control (p < 0.05). Exposure to sodium bicarbonate solution and a camomile solution, for 1 and 3 minutes, was not significantly lethal to oral biofilms nor to any microorganism evaluated, regardless of whether they were concentrated or diluted. We do not recommend the use of H2O2 but suggest using the camomile solution and NaF 0.02 percent in a rational manner for cleaning the infant's mouth.     

Enterococcus faecalis adhesin, Ace, mediates attachment to particulate dentin

An enzyme linked immunosorbent assay was developed to assess E. faecalis adhesion to particulate dentin. E. faecalis, OG1RF, which expresses the collagen binding protein (Ace+), and a derivative of OG1RF, TX5256, deficient in the collagen binding protein (Ace-) were grown at 46 degrees C, necessary for in vitro expression of Ace, and at 37 degrees C. E. faecalis binding to dentin was measured at 0, 15, 30, 60, 120, and 360 minutes. Compared to TX5256 and OG1RF grown at 37 degrees C, OG1RF grown at 46 degrees C adhered significantly better at all time points except 15 minutes (p < 0.001) exhibiting maximum binding at 120 minutes (17.4% of a positive control). Type I collagen at 100 microg/ml inhibited dentin binding by OG1RF grown at 46 degrees C in both competition (p < 0.005) and displacement assays (p < 0.046). Immunoaffinity purified anti-Ace IgG at 200 microg of protein inhibited adhesion of OG1RF grown at 46 degrees C to dentin.     

Susceptibility of Enterococcus faecalis biofilm to antibiotics and calcium hydroxide

The purpose of this study was to investigate the antimicrobial efficacy of six groups of antibiotics and calcium hydroxide against Enterococcus faecalis biofilm in a membrane filter model. Two-day-old E. faecalis (ATCC 29212) biofilm was exposed to ampicillin, co-trimoxazole, erythr omycin, oxytetracycline, vancomycin, vancomycin followed by gentamicin, Ca(OH)(2), and phosphate-buffered saline (control). After 1 h of exposure, the antimicrobial activity was neutralized by washing each disc five times in PBS, and then the colony-forming units of the remaining viable bacteria on each disc were counted. The results revealed that only erythromycin, oxytetracycline and Ca(OH)2 showed 100% biofilm kill. An ANOVA with a Bonferroni post hoc test (P < 0.05) detected significant differences among the test agents, except in the ampicillin group versus the co-trimoxazole group. It is concluded that erythromycin, oxytetracycline and Ca(OH)2 are 100% effective in eliminating E. faecalis biofilm, whereas ampicillin, co-trimoxazole, vancomycin, and vancomycin followed by gentamicin are ineffective.     

Low surface tension calcium hydroxide solution is an effective antiseptic

The antimicrobial effects of a saturated calcium hydroxide solution, and in combination with 10% and 20% detergent, were evaluated on Streptococcus faecalis. Strepto-coccus sanguis, Streptococcus mutans, Streptococcus salivarius, Neisseria sp., diphlheroid, Staphylococcus aureus, Lactobacillus sp., Staphylococcus epidermidis, Bacillus suhtilis and Candida albicans. The saturated calcium hydroxide solution was effective against only four of the 11 microorganisms studied over a 60-min exposure time. The calcium hydroxide solutions con-taining detergent killed all 11 test organisms over a 30-min exposure time. This difference was statistically significant (P<0.01). No statistically significant difference in antimicrobial action was found between the 10% and 20% detergent calcium hydroxide solutions (F>0.01). However, the low surface tension (46.5 × 10^?3 Nm^?1) and high pH (10.8) of the calcium hydroxide solution with 20% detergent establish it as the more effective solution.     

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.     

Photodynamic therapy for endodontic disinfection

The aims of this study were to investigate the effects of photodynamic therapy (PDT) on endodontic pathogens in planktonic phase as well as on Enterococcus faecalis biofilms in experimentally infected root canals of extracted teeth. Strains of microorganisms were sensitized with methylene blue (25 microg/ml) for 5 minutes followed by exposure to red light of 665 nm with an energy fluence of 30 J/cm2. Methylene blue fully eliminated all bacterial species with the exception of E. faecalis (53% killing). The same concentration of methylene blue in combination with red light (222 J/cm2) was able to eliminate 97% of E. faecalis biofilm bacteria in root canals using an optical fiber with multiple cylindrical diffusers that uniformly distributed light at 360 degrees. We conclude that PDT may be developed as an adjunctive procedure to kill residual bacteria in the root canal system after standard endodontic treatment.     

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 susceptibility of oral Candida albicans strains to different pH levels and calcium hydroxide saturated aqueous solution

Candida albicans is present in the oral cavity and in the whole digestive tract of humans and other animals, being frequently related to endodontic treatment failure. The present study determined the incidence of C. albicans in the oral cavity and the susceptibility of isolates to different pH values and saturated calcium hydroxide aqueous solution at pH 12.5. Sixty-five patients attending the Endodontic Clinic at the Sagrado Cora??o University participated in the study. The collected samples were cultivated in selective media for C. albicans and the isolates were tested in terms of resistance to both alkaline pH and saturated aqueous solution of calcium hydroxide. In relation to time variables, yeast viability was assessed by the Sabouraud's agar culture and fluorescein diacetate and ethidium bromide fluorescent staining method. Results from the different pHs and experimental times, including those from different techniques measuring fungal viability, were compared using the chi-square and Fisher's exact tests (α=0.05). The yeasts became completely inviable after 48 h of contact with the calcium hydroxide solution. On the other hand, when exposed to the alkaline culture broth, the yeasts were found to be viable at pHs 9.5 and 10.5 for up to 7 days. In conclusion, C. albicans can only be completely inhibited by direct contact with saturated calcium hydroxide aqueous solution after 48 h of exposure.     

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.