Tecnai 200kv electron microscope

To label PilY1 protein tagged with FLAG, exponentially growing M. xanthus cells in filtered CYE were gently spotted on carbon coated gold grids and allowed to settle for 20 min. The adhered cells fixed with 0.1% glutaraldehyde in PBS for 10 minutes. The grids subsequently were placed on drops: PBS–50 mM NH₄Cl (5 min), PBS–1% bovine serum albumin (5 min), the primary antibody (αFLAG, anti-DDK rabbit polyclonal, OriGene Technologies Inc., Rockville, MD, USA) diluted 1/100 in PBS–1% BSA (1h), three washes in Aurion incubation buffer (2 min each), Prot G (Aurion, 10-nm-diameter gold particles) diluted 1/40 in incubation buffer (30 min), incubation buffer (3×3 min), PBS (3×5 min), glutaraldehyde 1% in PBS (10 min), dH₂O (3×5 min). Grids without further staining were dried on filter paper and the samples were analyzed using a Tecnai 200KV electron microscope (Thermo Fisher Scientific), and digital acquisitions were made with a numeric camera (16 megapixel, CMOS, Oneview, Gatan).

TEM was performed on a FEI Tecnai 200 kV electron microscope (Thermo Fischer, Hillsboro, OR, USA) operating at 100 keV. The samples for the TEM observation were prepared by directly depositing the as-spun fibers onto the copper grids.

To observe bacterial pili, 5 μl drops of exponentially growing M. xanthus cells in filtered CYE medium were placed directly on glow discharged carbon coated grids (EMS) for 3 minutes. The grids were then washed with ten drops of distilled water. Grids without further staining were dried on filter paper and the samples were analyzed using a Tecnai 200KV electron microscope (Thermo Fisher Scientific) and digital acquisitions were made with a numeric camera (16 megapixel, CMOS, Oneview, Gatan).

Surface-associated T4P in S. sanguinis Δfim were visualised by TEM after negative staining as follows. Bacteria were grown in THTH until OD₆₀₀ reached 0.8, adsorbed for 3 min to glow-discharged carbon-coated grids (EMS), and fixed 5 min in 2% glutaraldehyde. The grids were cleaned by floating them sequentially 10 times on drops of pilus buffer (20 mM Tris, pH 7.5, 50 mM NaCl), and then stained for 2 min with 2% aqueous uranyl acetate. Stain solution was gently drained off the grids, which were air-dried before visualisation using a Tecnai 200 KV electron microscope (Thermo Fisher Scientific). Digital image acquisition was made with a 16 megapixel, CMOS, Oneview numeric camera (Gatan).