Evaluating the Effects of Disinfectants on Bacterial Biofilms Using a Microfluidics Flow Cell and Time-Lapse Fluorescence Microscopy.

A commercially available microfluidics flow cell was utilized together with widefield fluorescence microscopy to evaluate the effects of disinfectants on bacterial strains. The flow cell's inner surface supports the formation of biofilms of numerous bacterial species. The modular setup of the flow cell accessories allows connection to syringes, pumps and collection vials, facilitating aseptic experiments in a controlled fluidics environment which can be documented with precisely timed microscopy imaging. The flow cell is inoculated with a suspension of bacteria in a nutrient medium and incubated for several days allowing bacterial cells to form a biofilm. Shortly before performing an assay, the biofilm is labelled with a dual-fluorescent DNA probe which distinguishes unharmed and damaged bacteria. Then a disinfectant sample (or control) is gently injected and time-lapse imaging is used for quantifying the course of bacterial biomass response. We use a simplified widefield microscopy method that allows intensive recording and quantification of time series of two-dimensional frames for tracking the course of disinfectant action on a variety of microbial strains. This procedure has potential for the rapid evaluation of novel products.

Role of Dilution Rate and Nutrient Availability in the Formation of Microbial Biofilms.

We revisited the mathematical model of the chemostat and examined consequences of considerably decreasing the concentration of limiting nutrient in the inflow for the growth of both the planktonic and biofilm cells in the chemostat tank (fermenter). The model predicts a substantially lower steady-state biomass of planktonic cells in response to decreasing inflowing nutrient concentration. Contrarily, the steady-state concentration of nutrient inside the fermenter is expected to remain the same, as long as the inflowing concentration does not fall below its value. This allows the biofilm cells to grow at a rate regulated only by the exchange rate of the medium (dilution rate). We maintained a strain of Enterococcus faecalis in a chemostat of our own design with limiting nutrient in the inflow set near saturation constant at three dilution rates (0.09, 0.28, and 0.81 h-1). The highest dilution rate was near the critical rate calculated by the model. The one-day total biofilm buildup was 21× larger and its estimated growth rate 2.4× higher at highest dilution rate than at the lowest one. This increased biofilm formation with increased dilution rates is in agreement with previously published data on pure and mixed continuous flow cultures.

Assessment of different dyes used in leakage studies.

The goal of this in vitro study was to identify the most suitable dye for endodontic dye leakage studies, which could be a further step towards standardisation. The root canals of 70 extracted, single-rooted human adult teeth were enlarged to apical size 50 using hand instruments. The teeth were divided into seven groups (n = 10 each), and all root canals were completely filled by injection with one of the following dyes: methylene blue 0.5% and 5%, blue ink, black ink, eosin 5%, basic fuchsin 0.5% and drawing ink. Transverse root sections from the coronal, middle and apical part of the roots were examined, and the percentage of the dentine penetrated by dye was evaluated by software-supported light microscopy. In addition, the range of particle size of drawing ink particles was evaluated. There were conspicuous differences in the relative dye penetration into the root dentine and the penetration behaviour in the different root sections (two-way ANOVA, both p < 0.0001). One dye (drawing ink) penetrated less into the root dentine compared with all the others (p <0.0001). The particle size of this agent (0.1-2 microm) corresponds best with the size range of a representative selection of 21 species of pathogenic endodontic bacteria. Compared to the other dyes tested, drawing ink appears to be superior for use in endodontic dye leakage studies. The penetration behaviour into the root dentine of all the other dyes tested might be one factor that limits the applicability of these dyes in dye leakage studies.

Antibacterial efficacy of MTAD final rinse and two percent chlorhexidine gel medication in teeth with apical periodontitis: a randomized double-blinded clinical trial.

INTRODUCTION: Clinical assessment of the efficacy of novel root canal disinfection protocols is an important focus in endodontic research. This randomized double-blinded study assessed the antibacterial efficacy of a final rinse with BioPure MTAD (MTAD) and intracanal medication with 2% chlorhexidine gel (CHX) in teeth with apical periodontitis. METHODS: Canals in 30 teeth (single-rooted and multi-rooted) were prepared by using 1.3% NaOCl, rinsed with MTAD or saline in random sequence, medicated with CHX for 7 days, irrigated with 1.3% NaOCl, and filled. Bacteriologic root canal samples were obtained by aspiration before (1A) and after (1B) canal preparation, after the final rinse (1C), after CHX was flushed (2A), and after final irrigation (2B). Bacteria were enumerated by epifluorescence-microscopy (EFM) by using 2 staining methods and by colony-forming-unit (CFU) counts after 14 days of incubation. RESULTS: Bacterial counts (EFM) in 1B were greater than 95% decreased from 1A. Low bacterial densities in 1B, 1C, 2A, and 2B did not differ significantly from each other. EFM counts were consistently higher than CFU counts. CONCLUSIONS: The final rinse with MTAD and medication with CHX did not reduce bacterial counts beyond levels achieved by canal preparation with NaOCl.

Characterization of an ex vivo model for the assessment of root canal disinfection.

Root canal bacteria in teeth with apical periodontitis were enumerated after extraction and incubation. Canals in 36 teeth were sampled after: S1, incubation for 2 hours (group A), 2 days (group B), 4 days (group C), and 6 days (group D); S2, subsequent incubation for 1 week; S3, canal disinfection; and S4, final incubation for 1 week. Bacterial concentrations were determined by culture (colony-forming unit [CFU]) and epifluorescence-microscopy (EFM) and compared by using pairwise and exact-permutation tests (p < 0.05). CFU counts were lower than EFM counts. CFU counts in S1 were higher in Gp(A) than in Gp(C) (p < 0.004) and Gp(D) (p < 0.02). EFM counts in S1 were higher in Gp(A) than in Gp(C) (p < 0.02). Both enumeration methods showed bacterial counts decreasing from S1 to S2 (p < 0.04). EFM was superior to culture in this ex vivo model. The indigenous flora survived incubation for 6 days, but the adverse effect of initial access would preclude testing of disinfection protocols that require two sessions.

Antibacterial efficacy of chlorhexidine gluconate intracanal medication in vivo.

The antibacterial efficacy of intracanal medication with 2% chlorhexidine liquid (CHX) was assessed in teeth with apical periodontitis. Canals in 22 teeth were instrumented at the first session, medicated with CHX, and reaccessed after 7 to 15 days. Bacteriological samples were aspirated at the first and second sessions, before (1A, 2A) and after (1B, 2B) canal instrumentation. Viable bacterial counts were obtained by culture (CFU) and microscopy using vital dyes. Microscopic counts were higher than CFU counts. Consistently high CFU counts in 1A samples (mean, 2 x 10(5) microL(-1) canal volume) decreased significantly (p < 0.0001) in 1B samples, increased significantly (p < 0.04) in 2A samples, and decreased in 2B samples to the level of 1B samples. Proportions of negative cultures followed the pattern of CFU counts. Intracanal medication with CHX did not reduce the bacterial concentration. Bacterial counts expressed per microliter canal volume added information beyond the counts per tooth as expressed in previous studies.

In vitro leakage associated with three root-filling techniques in large and extremely large root canals.

This study assessed the apical leakage of ultrasonically condensed root fillings in extremely large canals, compared to cold lateral condensation and thermoplastic compaction. Ninety single-rooted teeth were used. In 45 teeth canals were enlarged to size 70 (large). The remaining 45 canals were enlarged to size 140 (extremely large). Each set of teeth was subdivided into three root-filling groups (n = 15): (1) cold lateral condensation (LC); (2) thermoplastic compaction (TC); and (3) ultrasonic lateral condensation (UC). Teeth in all six subgroups were subjected to drawing ink penetration, cleared, and evaluated for linear apical dye leakage. Significantly deeper dye penetration (p < 0.04, Wilcoxon rank-sum test) was observed for LC than for UC. TC did not differ significantly from LC and UC. Dye penetration was significantly deeper (p < 0.0001) in canals enlarged to size 140 than to size 70, independent of root-filling method. Apical leakage associated with ultrasonically condensed root fillings was less than that with cold lateral condensation. It was consistently greater in extremely large canals than that in large ones.