Real-Time Imaging and Quantification of Bioluminescent Bacteria in Root Canals In Vitro

Abstract

The detection of microorganisms in root canals is generally limited to qualitative or semiquantitative methods. We describe a new and nondestructive in vitro method to quantify root-canal bacteria over sequential treatment procedures using real-time imaging in conjunction with the bioluminescent reporter strain Pseudomonas fluorescens 5RL. Induced bacterial photon emission can be monitored by sensitive optical photonic imaging and luminometry, providing images as well as spatial and quantitative measurements. Bioluminescence imaging and luminometry determined that the lower limit of detection of bacteria in root canals occurred between 2 x 10(2) and 2 x 10(3) cells, with high correlation between cell counts and detection devices (r > or = 0.981). A preliminary study assessed the efficacy of sequential irrigation procedures to remove 5 x 10(6) bacteria from root canals (n = 5; apical size 60) using a 28-gauge, endodontic needle positioned 1 mm from working length; 9.2% +/- 3.1% and 8% +/- 3.6% of bacteria remained after 3 and 6 ml irrigation, respectively (p = 0.03), corresponding to approximately 4 x 10(5) bacteria remaining after 6 ml. This method can be used to study the efficacy of sequential endodontic treatment procedures in removing bacteria from root canals.

Time-dependent Antibacterial Effects of the Self-Adjusting File Used with Two Sodium Hypochlorite Concentrations

“…Thus, the 4-minute and the 6-minute approaches resulted in irrigant volumes that were 10 mL and 20 mL larger than the 2-minute approach, respectively. A previous study using a similar in vitro model suggested that, although the volume of irrigant can influence bacterial elimination (26,27), there may exist a point of saturation, above which the increase in volume will no longer affect bacterial elimination (28). Therefore, how the increased volume after each time interval influenced the results remain to be determined.…”

The Effect of Needle-insertion Depth on the Irrigant Flow in the Root Canal: Evaluation Using an Unsteady Computational Fluid Dynamics Model

“…Because the needle is positioned further away from WL, the increased number of vortices causes a delay in the irrigant replacement. A higher volume of irrigant, which has been shown to affect the irrigation efficiency (20), can be translated to an increased duration of irrigation when the flow rate is constant according to the definition of flow rate (Q): Q ¼ DV Dt , where DV is the volume of fluid crossing the control surface (in this case delivered through the needle) during the specified time period (Dt) (21). An increased duration of irrigation is expected to improve irrigant exchange in all cases, regardless of needle position, magnifying any existing difference.…”

Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection

“…The method provides an alternative to traditional in vitro culture methods using paper point sampling and quantitative culture, and further supports previous reports about the difficulties of completely removing bacteria from root canals. Sedgley et al [51] developed a bioluminescent model of root canal infection using Pseudomonas fluorescens 5RL which is a strain containing a lux CDABE plasmid that is inducible with salicylate. They determined the correlation between bioluminescent signal and extracted CFUs.…”