200 mesh copper em grids

Blocks were transferred to aluminum cryo-sectioning pins (Leica) and quickly plunge-frozen in liquid nitrogen. Thin cryo-sections (80 nm) were cut at −100°C with an EM-UC6/FC7 cryo-ultramicrotome (Leica) using a cryo-diamond knife (Diatome). Cryo-sections were removed from the knife with 2.3 M sucrose using a wire loop and transferred to formvar/carbon-coated, plasma cleaned, 200-mesh copper EM grids (Proscitech). Grids were stored in an airtight container on sucrose droplets at 4°C. To stain, grids were floated face down on 2% gelatin for 30 min at 37°C before washing in PBS (3 min × 2 min) and staining with 2% uranyloxalicacetate, pH 7 (5 min, room temperature) and methyl cellulose–uranyl acetate pH 4 on ice (10 min). Grids were looped out, drained, and allowed to dry. Samples were imaged with a Tecnai G2 Spirit electron microscope (FEI Company) operated at 100 kV at Adelaide Microscopy, the University of Adelaide, South Australia.

Xen41 cells were prepared essentially as described for Xen14, then processed for TEM using Procedure 4 (Table 1) with either 1 h fixation or overnight fixation followed by post fixation in 1% osmium tetroxide for 1.5 h on ice.
Sections of Xen14 and Xen41 embedded in resin were cut to 1 μm using a glass knife, stained with 1% toluidine blue containing 1% borax and viewed under a light microscope at 400× magnification to identify stained bacteria. Ultrathin sections were then cut to 90 nm with an ultramicrotome EM-UC6 (Leica) using a diamond knife (Diatome) and placed on 200-mesh copper EM grids (Proscitech). Sections were sequentially stained with uranyl acetate (4% in distilled H₂O) and Reynolds lead citrate for 10 min each, with three washes in distilled water in-between each stain. Sections were then viewed on a Tecnai G2 Spirit (FEI Company, Hillsboro, OR, USA) Transmission Electron Microscope operated at 100 KV at Adelaide Microscopy, The University of Adelaide.