2.5 Microscopy
Routine microscopic observations were performed using an Optiphot microscope (Nikon) and an S4E stereomicroscope (Leica). The morphology of the bacterial cells was observed using a SEM (JSM-6340F; JEOL). For scanning electron microscopy, cells were fixed in 0.1 M cacodylate buffer (pH 7.4) containing 4% (wt/vol) paraformaldehyde and 4% (wt/vol) glutaraldehyde at 4°C for 2 h, washed with 0.1 M cacodylate buffer (pH 7.4) once, fixed again in 0.1 M cacodylate buffer (pH 7.4) containing 2% (wt/vol) glutaraldehyde at 4°C for overnight, washed again in 0.1 M cacodylate buffer (pH7.4), and fixed once again in 1% (wt/vol) tannic acid in 0.1 M cacodylate buffer (pH 7.4) at 4°C for 2 h. Fixed cells were washed four times in 0.1 M cacodylate buffer (pH7.4), and post-fixed with 2% (wt/vol) osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) at 4°C for 3 h. Samples were dehydrated in an ethanol series [50%, 70%, 90%, and 98% (vol/vol)] each for 30 min, transferred into t -butyl alcohol, freeze-dried under a vacuum, and coated with a thin layer of osmium by using an osmium plasma coater (NL-OPC80NS; Nippon Laser and Electron Laboratory). The SEM was also used to observe the surfaces and cross-sections of corroded Fe0 foils.