Use of a Microcentrifuge for Preparation of Isolated Mitochondria and Cell Suspensions for Electron Microscopy

The special problems of preparing suspensions of cell fractions such as mitochondria for electron microscopy are largely eliminated once the fixed organclles are gathered into a cohesive pellet. Thesc pellets may then be handled in the manner conventional for blocks of tissue (1, 2). The use of very small samples (e.g., the mitochondrial yield from 4 mg of rat liver) has been made possible by the use of a small, high speed centrifuge only recently available. Customary glass or cellulose nitrate (Lusteroid) centrifuge tubes and related apparatus have been awkward to use for the preparation of samples for electron microscopy. Small conical glass tubes may require specially made adapters to fit them to heads for larger tubes. In addition, until the mitochondria are aggregated into a cohesive pellet, changes of solution must be tediously accomplished by repeated ccntrifugation, decantation, and resuspension of the sample in the new solution. In order to handle the final pellet as a tissue block, the packing must bc tight enough to minimize erosion of the sample during the subsequent solution changes. When cellulose nitrate tubes are used, packing of small samples is inefficient, because the smallest of thcse tubes have rounded bottoms. The flat or concavc pellets embedded in these tubes present a broad face to the microtome knife. Finally, where the amount of material available for clcctron microscopy is greatly limited, as in correlatcd biochemical and morphological studies, this sort of pellet may be useless (3). What is needed, then, is a method of accumulating a small volume of mitochondria (or other dispersed particulates) into a near cylindrical or conical pellet whose shape may be maintained from fixation through hardening of the embedding medium and whose long axis may be then oriented perpendicular to the microtome knife. This would minimize wastage through trimming of the block of embedding medium and would be especially advantageous when many sections of a relatively small volume are desired.