A1 and ti e

The bacteria cells were cultured in 100 mL LB medium for 24 h in the presence of 20 mg/L of Pb (II) and collected by centrifuge at 8000 × g for 10 min, followed by suspension in saline buffer. Three different staining dyes were sequentially added to the cell suspension to stain the DNA (DAPI Nucleic Acid Stain, Molecular probes, Invitrogen), EPS (Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate, Molecular Probes, Invitrogen), and Pb (II) (Leadmium Green AM Dye, Molecular Probes, Invitrogen). The samples were analyzed with a CLSM (Nikon A1 and Ti-E) equipped with a Plan Apo VC × 60 objective lens (NA 1.40, Nikon).

Dead and whole cells were detected with propidium iodide (PI) and Hoechst 33342 (Dojindo, Kumamoto, Japan) 3 or 7 days after the injection [16] (link). Endplates were removed with a scalpel blade, and NP tissues were harvested using a biopsy punch. Harvested NP tissues were stained with 4 µM PI and 4 µM Hoechst 33342 for 1 h. PI emits red fluorescence in dead cells, whereas Hoechst 33342 emits blue fluorescence in all cells, regardless of live or dead status. The stained NP tissues were washed with phosphate-buffered saline (PBS) and examined using a confocal laser scanning microscopy system (Nikon A1 and Ti-E, Tokyo, Japan) equipped with a Plan Fluor 20× objective lens (N.A., 0.45; Nikon). The number of dead and all cells in each NP tissue was measured from >3 randomly chosen stacks, each representing a 100-µm projection at a 5-µm interval. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for cell number quantification [12] (link), [13] (link). All the experiments were performed on 6 discs from each treatment group and time point.