Scanning transmission electron microscopy.

The appropriate strains were grown to early exponential phase anaerobically in 400 mL of BHI. Cultures were centrifuged for 10 min at 4,500 rpm, and pellets were washed once in 1 M PBS. Pellets (~100 μL) were mixed with equal volumes of fixative buffer consisting of 4% mass spectrometry-grade paraformaldehyde and 0.1 M sodium cacodylate. After 30 min, cells were pelleted and mixed with equal volumes fresh fixative buffer for 1 h at room temperature. Samples were then rinsed with 0.1 M sodium cacodylate buffer, postfixed in osmium tetroxide buffered with 0.1 M sodium cacodylate for 2 h, and then washed to remove osmium tetroxide. Samples were then progressively dehydrated with increasing concentrations of ethanol (50, 70, 90, and 100%) and twice with 100% acetone. Bacteria were then embedded in 50:50 acetone-resin (Epon) and incubated rotating for 4 h. Incubation was next performed using 20:80 acetone-resin. After drying for 2 h, the samples were placed in a 100% resin gelatin capsule under light vacuum for 2 h. Finally, samples were placed in fresh resin molds and incubated at 60°C overnight. Resin blocks were sectioned at 80 nm using a Leica Ultracut 6 Microtome and stained using uranyl acetate and Reynolds lead citrate stains. Samples were imaged with an FEI Tecnai G2 F20 Twin transmission electron microscope at 200 kV.