Evaluation ofFreeze-Substitut ion andConventional Embedding Protocols forRoutine Electron Microscopic Processing ofEubacteria

oftwoisogenic derivatives ofEscherichia coli K-12asrepresentative gram-negative eubacteria andintotheRNA andpeptidoglycan ofBacillus subtilis strains 168andW23 as representative gram-positive eubacteria. Radiolabeled bacteria wereprocessed forelectron microscopy by conventional methods withglutaraldehyde fixation, osmiumtetroxide postfixation, dehydration ineither a graded acetone orethanol series, andinfiltration ineither SpurrorEpon812resin. A second setofcells were simultaneously freeze-substituted byplunge-freezing inliquid propane, substituting inanhydrous acetone containing 2% (wt/vol) osmiumtetroxide, and2% (wt/vol) uranyl acetate, andinfiltrating inEpon812. Extraction ofradiolabeled cell components wasmonitored byliquid scintillation counting atallstages of processing toindicate retention ofcelllabels. Electron microscopy wasalsousedtovisually confirm ultrastructural integrity. Radiolabeled nucleic acids andwail components wereextracted bybothmethods. In conventionally embedded specimens, dehydration wasparticularly damaging, withethanol-dehydrated