I. loss of lipids during preparation of amoebae for electron microscopy

Abstract

1. 1. Amoebae were fixed in glutaraldehyde, potassium permanganate or osmium tetroxide, alone or in combination, and extracted with ethanolic solutions, propylene oxide and embedding solution as in preparation for electron microscopy. The lipid that was extracted by each solvent was measured chemically (ester and phosphorus analyses) and by radioactive analysis using amoebae grown in the presence of [ 3H]-palmitic acid. The extracted lipids were fractionated by silicic acid chromatography into neutral lipids and phospholipids and the fatty acid composition of each was determined. 2. 2. After fixation in glutaraldehyde, the lipids were unchanged and were almost completely extracted by ethanol during the dehydration procedure. After perman-ganate fixation, all of the neutral lipid and about 25% of the phospholipid were extracted by the fixative and ethanol. Following fixation in osmium tetroxide, most of the neutral lipid and some of the phospholipid were extracted by ethanol and the embedding medium. Osmium tetroxide destroyed the unsaturated fatty acids. 3. 3. It is concluded that autoradiographic studies of lipid metabolism at the electron microscopic level are not possible if the usual methods of fixation and dehydration are employed. Also, structures visualized in the cell after fixation in glutaraldehyde alone probably do not contain lipid at the time of observation.