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Effect of fixatives on the unstable functional states of mitochondria was studied. The energized configuration of the crista! membrane of beef heart mitochondria can be maintained and visualized by the electron microscope if fixatives are introduced before the oxygen supply is exhausted. Since the exhaustion of the available oxygen supply is completed in 5-20 seconds, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is most effective for freezing the configurational state of crista! membrane. Fixation with glutaraldehyde appears to be complete within 1-2 seconds even at 0°C. Osmium tetroxide alone can also "freeze" the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). When the level of glutaraldehyde or osmium tetroxide is respectively too low or too high, the mitochondria will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering measurements which provide an independent method for monitoring configurational changes in mitochondria confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration.

Ultrastructure and functional states of mitochondria. Effect of fixatives on the stabilization of unstable configuration.