P. aeruginosa PAO1 forms BLS within the ASM+. After 48 h of growth at 37°C under 20% EO2/static conditions, PAO1/pMRP9-1 developed BLS that were confined to the ASM+ and not attached to the surface of the microtiter plate. The composition of the ASM+ and the bacterial inoculation are described in Methods. The gelatinous mass containing the BLS was visualized in situ by CLSM. (A) CLSM micrograph of the PAO1/pMRP9-1 BLS; magnification, 10X; bar, 200.00 nm. (B) 3-D image analysis revealing the architecture of the BLS shown in (A); box, 800.00 pixels (px) W x 600 px H; bar, 100 px. (C) CLSM micrograph of the well bottom after the removal of the gelatinous mass showing no attached bacteria or biofilm (the scattered fluorescence observed is due to autofluorescing debris).