Cem 902 electron microscope

To image H. neptunium cells, 10 μl of early exponential cell cultures were applied onto glow-discharged electron microscopy grids (Formvar/Carbon Film on 300 Mesh Copper; Plano GmbH, Germany) and incubated for 1 min at room temperature. The grid was manually blotted with Whatman filter paper to remove excess liquid. Subsequently, the cells were negatively stained for 5 sec with 5 μl of 1% uranyl acetate. After three washes with H₂O, the grids were dried and imaged in a 100 kV JEM-1400 Plus transmission electron microscope (JEOL, USA). To image BacA polymers, purified BacA-His6 was dialyzed against low-salt buffer (50 mM HEPES pH 7.2, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM ß-mercaptoethanol) for 16 hr. The protein was spotted onto carbon-coated grids and allowed to settle for 2 min. The grids were blotted dry, stained with 1:2 diluted supernatant of saturated 2% uranyl acetate (in H₂O) for 1 min, dried and imaged using a Zeiss CEM902 electron microscope, operated at 80 kV and equipped with a 2048 × 2048 pixel CCD camera. Image processing was carried out using Photoshop CS2 and Illustrator CS5 (Adobe Systems, USA).

The gastrocnemius muscle was fixed for 5 min with a solution consisting of 2.5% glutaraldehyde, 1% paraformaldehyde, and 0.01% sodium azide in 0.05 M sodium cacodylate at pH 7.2 and then removed. The specimens were further fixed in the same solution overnight, followed by post-fixation in 1% OsO₄ in 0.1 M sodium cacodylate, dehydration, and infiltration with epoxy resin (Agar 100), in accordance with routine procedures. Ultrathin sections were cut with diamond knives in a Reichert Ultracut E microtome and contrasted with lead citrate and uranyl acetate before examination in a Zeiss CEM 902 electron microscope.