The study of steady-state concentrations of internal solutes of mitochondria by rapid centrifugal transfer to a fixation medium

Reliable measurements of the distribution of solutes between mitochondria and their surrounding medium depends ideally on the rapid isolation of mitochondria from their medium. Moreover, it is essential that the solute composition of mito- chondria should not change during this process. A method is described in this paper which deals with this problem. The mitochondria are centrifuged through a layer of silicone fluid immiscible with water, of a density intermediate between that of the particles and that of the medium. A high-density aqueous 'fixative' beneath the silicone can be used to stabilize labile compounds. Particles centrifuged in this manner carry with them an adherent film of their incubation medium. The amount of this adherent fluid can be measured by the inclusion of a radioactive high molecular (20 000-50 000) poly- saccharide, such as inulin or polyglucose.