Observation of Pseudomonas aeruginosa biofilm with confocal laser scanning microscope

Confocal laser scanning microscope (CLSM) is a new type of microscope, which permits non-invasive optical sectioning of biomaterials by reducing out-of-focus haze. A biofilm of Pseudomonas aeruginosa was developed on the silicon disks using a modified Robbins device and used to visualize hydrated living biofilm with CLSM. In order to examine optimum staining agents and conditions, acridine orange, fluorescein isothiocyanate (FITC), FITC-conjugated concanavalin A (FITC-ConA), Evans blue, safranine and rhodamine-conjugated concanavalin A were used in three different medium (pH 4.5, pH 7.5, pH 9.5). Only extracellular matrix was stained as a net-like structure for FITC-ConA and only bacteria for acridine orange and safranine. Although the staining patterns with FITC and Evans blue were affected markedly by the medium pH, those with other staining agents were not affected significantly. It was considered that the staining characteristics specific for each agent and changes of staining pattern by pH were probably due to relative differences among matrix, bacteria and staining agents in their electrostatic charges. In addition to sagittal sectioning images of P. aeruginosa biofilm, clear doubles staining images, which differentiates bacteria from matrix in the identical material, could be obtained with the combination of safranine and FITC-ConA.