Discrete localization of stain in pericanalicular granules was found in 10 µ frozen sections of formol-phosphate-sucrose-fixed liver stained by the Gomori acid phosphatase technique and examined in the light microscope. The staining patterns, before and after treatment with Triton X-100 and lecithinase, were identical with those previously reported for formol-calcium-fixed material treated in the same way, and it can be assumed that the stained granules are identical with "lysosomes." Examination in the light microscope of the staining patterns and lead penetration in fixed blocks and slices of various dimensions showed nuclear staining and other artefacts to be present, produced by the different rates of penetration of the various components of the staining medium into the tissue. A uniform pericanalicular staining pattern could be obtained, however, with slices not more than 50 µ thick, into which the staining medium could penetrate rapidly from both faces. The staining pattern produced in 50 µ slices was the same both at pH 5.0 and pH 6.2, and was not altered by subsequent embedding of the stained material in butyl methacrylate. Electron microscopy showed the fine structure of fixed 50 µ frozen slices to be well preserved, but it deteriorated badly when they were incubated in the normal Gomori medium at pH 5.0 before postfixing in osmium tetroxide. After incubation in the Gomori medium at pH 6.2, the detailed morphology was substantially maintained. In both cases lead phosphate, the reaction product, was found in the pericanalicular regions of the cell, but only in the vacuolated dense bodies and never in the microbodies. Not every vacuolated dense body contained lead, and stained and unstained bodies were sometimes seen adjacent to each other. This heterogeneous distribution of stain within a morphologically homogeneous group of particles is consistent with de Duve's suggestion (9) that there is a heterogeneous distribution of enzymes within the lysosome population. It is concluded from these investigations that the vacuolated dense bodies seen in the electron microscope are the morphological counterparts of the "lysosomes" defined biochemically by de Duve.

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A study has been made of the preservation of fine structure, phospholipids, and the activity of acid phosphatase and esterase in rat liver fixed in various solutions containing 4 per cent formaldehyde. Examination of methacrylate-embedded preparations shows that calcium-containing fixatives result in poor preservation of fine structure, whereas veronal-treated or phosphate-buffered formalin gives excellent results if the tonicity of the solutions is suitably adjusted by addition of sucrose. Formol-phosphate, to which Versene has been added, causes deterioration of cellular morphology. Phospholipids are retained almost quantitatively in tissue fixed in formol-calcium, and in phosphate-, collidine-, or triethanolamine-buffered formalin. About 50 per cent of the activity of acid phosphatase and esterase are preserved after 24 hours exposure to these fixatives at 0–2°C, and the distributions of the enzymes and of phospholipids, as judged by cytochemical staining results, are not altered by any of these formalin solutions. Consideration of the morphological and biochemical integrity of the fixed tissue suggests that 4 per cent formaldehyde, buffered at pH 7.2 with 0.067 M phosphate, and containing 7.5 per cent sucrose, is the most suitable of the fixatives for combined cytochemical staining and electron microscopical studies.