A specially designed high-speed blendor and glass beads have been used to disintegrate yeast cells. The method enables large quantities of cells to be fragmented quickly at low temperature, and cell-free mitochondrial particles to be prepared in high yield. The particles are isolated in a sucrose-Tris-EDTA medium and extensively refractionated in the same medium. The success of the fractionation is dependent upon the presence of the Tris buffer, as the latter prevents the aggregation of the particulate material. Two morphologically and enzymatically different particle types have been obtained: a heavy fraction corresponding to mitochondria in size and internal organization, and a light fraction consisting of vesicular, single-membrane particles of a smaller size. The light particles oxidize DPNH and succinate, but do not oxidize pyruvate-malate, and lack the capacity for phosphorylation. The heavy particles oxidize pyruvate-malate as well as the citric acid cycle intermediates, although their α-ketoglutaric dehydrogenase activity is low. Oxidation by the heavy particles is coupled to phosphorylation, and P/O ratios of about 1.5 have been obtained. Lactic acid dehydrogenase is also present in the heavy fraction, and lactate is oxidized with a P/O ratio of about 0.7.

Vegetative cells of Saccharomyces cerevisiae were fixed with potassium permanganate followed by uranyl nitrate, embedded in methacrylate, and studied in electron micrographs of thin sections. Details of the structure of the cell wall, cytoplasmic membrane, nucleus, vacuole, and mitochondria are described. Cell membranes, about 70 to 80 A thick, have been resolved into two dense layers, 20 to 25 A thick, separated by a light layer of the same dimensions, which correspond in thickness and appearance to the components of the "unit membrane" as described by Robertson (15). The cell wall is made up of zones of different electron opacity. Underlying the cell wall is the cytoplasmic membrane, a sinuous structure with numerous invaginations. The nucleoplasm, often of uneven electron opacity, is enclosed in a pair of unit membranes in which nuclear pores are apparent. The vacuole, limited by a single unit membrane, is usually irregular in outline and contains some dense material. Rod-shaped mitochondria, 0.4 to 0.6 µ in length and 0.2 to 0.3 µ in diameter, are smaller in size, but similar in structure to some of those described in plant and animal cells. Attempts to use osmium tetroxide as fixative were unsuccessful, a result similar to that obtained by other workers. It is suggested that yeast cells are impermeable to osmium tetroxide, except when grown under specific conditions.