Real-time imaging and quantification of bioluminescent bacteria in root canals in vitro

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.