Cryo holder model 626

The vegetative cells and spores from B. subtilis, S. violaceoruber, A. chroococcum, M. xanthus and A. cylindrica were prepared as described above; where necessary, culture volumes were doubled or tripled to obtain a higher biomass. The samples were resuspended in phosphate-buffered saline (PBS) supplemented with 30 % dextran (D1662 from Sigma-Aldrich, St Louis, MI, USA). They were then cooled under high pressure (2000 bar) to liquid nitrogen temperature (−196 °C) within milliseconds using an EM-PACT2 machine (Leica Microsystems, Wetzlar, Germany). These conditions prevent the formation of ice crystals, which can damage cellular structures [50 (link)]. The frozen samples were then cut into thin slices (50 nm) in a cryo-ultramicrotome UC6 FC6 (Leica Microsystems, Wetzlar, Germany) and placed on a R3.5–1 holey carbon EM grid (Quantifoil, Großlöbichau, Germany). Samples prepared in this way were then loaded into a Tecnai F20 transmission electron microscope (Thermo Fisher Scientific, Waltham, MA, USA) on a cryo-holder model 626 (Gatan, Pleasanton, CA, USA) keeping the sample temperature low (−180 °C) and analysed by the low-dose method at 200 kV acceleration voltage.

Cryosamples were prepared immediately after incubation with and without metal salts using a Vitrobot Mark IV (Thermo Fisher Scientific). Teflon sheets were used between the blotting pads and blotting paper to reduce contamination of the pads. 4 μl of sample was applied to freshly glow discharged lacey carbon grids (300 mesh copper grids, C269/C TAAB), blotted and plunged automatically into liquid ethane. Freezing conditions were set to: blotting force-1, blotting time 2 s, drain time 1 s and 5 s wait time with conditions in the sample preparation chamber set to 100% humidity and room temperature. Samples were stored under liquid nitrogen until imaging. Cryo-EM was performed using a Tecnai F20 200 kV electron microscope (Thermo Fisher Scientific). Samples were loaded into the EM using a cryoholder model 626 (Gatan). Imaging was carried out under low dose conditions, with a defocus value of −5 μm. Images were acquired using a CMOS F816 camera (TVIPS), using 8 kb2 settings.