Fluoview clsm

Sterile plastic cover slips (18 x 18 mm) placed in 12 well mutidishes (Nunc) were inoculated with 1.5 ml culture of K. pneumoniae (1 × 10⁸ CFU/ml) and 1.5 ml nutrient broth and incubated at 37 °C. Each day, spent medium was removed and replaced with fresh nutrient broth. On 3^rd and 7^th day, the cover slips were washed thrice with 0.85 % NaCl, followed by treatment with 20 units/ml of either phage or bacterial depolymerase for 1 h at 37 °C. Thereafter, the cover slips with untreated and treated biofilms were stained with 300 mg/ml calcofluor white (Sigma) [calcofluor white binds to the sugar residues in the biofilm polysaccharide and stains them blue] and 0.1 % Syto 62 (Molecular Probes) [Syto 62, a nucleic acid staining dye stains the bacterial cells red] for 15 min each. These were then examined under a Inverted Olympus Fluoview CLSM (FV 1000, Olympus America Inc. NY, USA). All images were obtained with a 20X lens. Multiple images were collected for each set of experimental conditions; a representative image is presented for each group in the results section. Image analysis was done using z-series image stacks from four randomly chosen spots of each biofilm. The ratio of cell biomass to polysaccharide was calculated by dividing the integrated red fluorescence density with integrated blue fluorescence density using ImageJ version 1.46r [57 (link)].

CLSM analysis was performed as previously described [3 (link)]. Cells were seeded onto multiwell glass-bottom dishes (Matsunami Industries, Osaka, Japan) at a density of 2.0 × 10⁴ cells/well in a final volume of 100 µL and incubated for 24 h at 37 °C under 5% CO₂. The expression of LAMP-1-RFP was observed with excitation wavelength of 555 nm and emission wavelength of 580 nm. The intracellular distribution of His16-Lyso was also determined by CLSM. LysoTracker Green (Life Technologies, Carlsbad, CA, USA) and Hoechst 33342 (Dojindo, Kumamoto, Japan) were used as an organelle marker for lysosomes and a nuclear stain, respectively. After complete adhesion, His16-Lyso were added and the cells were incubated for 24 h. The culture medium was replaced with fresh medium, and Hoechst 33342 was added to the cells and incubated for 1 h. After two washes with PBS, fresh medium containing 500 nM LysoTracker Green was added, and the cells were incubated for 2 h. The intracellular distribution of His16-Lyso was observed and fluorescence images were acquired using a FluoView CLSM (Olympus, Tokyo, Japan).