High-Pressure Freezing of C. elegans with E. coli OP50

C. elegans worms, including E. coli OP50 bacteria, were scraped on to the high-pressure freezing (HPF) specimen carriers. Bacteria served as a filler, minimizing water content and facilitating freezing [46 (link)]. Worms on the carriers were frozen in the high-pressure freezer (Leica EM PACT2). Frozen samples collected on metal carriers were transferred under liquid nitrogen into a pre-frozen cryotube containing 1 ml freeze-substitution solution (2% OsO4 in 100% acetone with 1% lecithin) and finally to a freeze substitution unit (Leica EMAFS) for processing. Samples embedded in Epon EmBed812 resin were ultrasectioned (80nm) with an ultramicrotome (Leica EM UC 6) and placed on copper mesh 300 previously coated with uranyl acetate and lead citrate. Sections were finally examined using a transmission electron microscope (JEOL JEM 2100-Plus 200kV).