Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy

Abstract

The loss of I4C-ethanolamine-and 3H-choline-labelled phospholipids from rat liver during preparation for electron microscopy by some less frequently used processing methods has been examined. Permanganate and formaldehydepotassium dichromate fixation followed by Araldite embedding were investigated and five procedures involving embedding in water-miscible methacrylates (GMA). These procedures included a conventional method of dehydration and embedding in GMA, a low-temperature GMA embedding method, dehydration with ethylene glycol, freeze drying and freeze substitution. These results are compared with those obtained after conventional tissue preparation (presented previously, Cope & Williams, 1969). Formaldehyde-potassium dichromate compared favourably with the conventional procedures for the preservation of both phosphatides, especially phosphatidyl ethanolamine. Permanganate fixation was much less effective. Severe loss of both phosphatides occured after freeze drying and freeze substitution in glutaraldehyde-alcohol. GMA is shown to be a more potent phospholipid solvent than ethanol under the conditions employed. Low-temperature embedding reduced the loss of phosphatidyl choline during embedding. Results obtained by scintillation counting were confirmed by grain counts on thick-section autoradiographs. No direct relationship between extraction and the electron-microscopic appearance of membranes was discernible. It is believed that membrane prominence is largely dependent upon the electron density of the surrounding cytoplasm rather than on the degree of phospholipid extraction.

Reevaluation of the effect of lysoyzme onEscherichia coli employing ultrarapid freezing followed by cryoelectronmicroscopy or freeze substitution

“…Employing a chemical fixation procedure, we then found that lysozyme per se affected E. coli, resulting in disintegration of the cytoplasm, whilst the membranes still surrounded the cytoplasm even in severely damaged cells, leading to the formation of cell ghosts. Fixation with aldehydes with or without postfixation with osmium tetroxide results in severe changes, including loss of lipids (Cope and Williams, 1969), ondulation of the cell membranes, and aggregation of cytoplasmic components (Kellenberger, 1991). Loss of both cytoplasmic and membrane material can be assumed to be enhanced in cells undergoing degenerative changes.…”

Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freeze-substitution

“…Over the last decade, freeze-substitution has been used most frequently for the examination of eucaryotic systems (10,12,13); it has rarely been used on procaryotic cells. For this reason, it has been difficult to ascertain the power and scope of freeze-substitution as a technique for the preservation of microbial systems.…”

Ultrastructural findings in human nonalcoholic steatohepatitis

“…Ultrastructural studies of fat droplets in NASH are useful because osmium tetroxide effectively preserves lipid-containing structures [32][33][34]. The fat droplet forms in or alongside the ER in conjunction with apolipoprotein B and adipophilin [35][36][37].…”