Osmium Tetroxide Postfixation Research Articles

Ultrastructural observations on lamellar and tubular membrane configurations in fungi.

A morphological study was made of membrane configurations in Penicillium viridicatum Westling and Trichothecium roseum Link, both members of the Fungi imperfecti. These membrane configurations (similar to previously described “myelin figures” and “mesosomes”) were observed after glutaraldehyde fixation and osmium tetroxide postfixation, but not after potassium permanganate fixation. Configurations with lamellar membranes occur in both P. viridicatum and T. roseum. In P. viridicatum, these configurations were found in the cytoplasm and in vacuoles, while in T. roseum they were found between the cell wall and plasmalemma and in the cytoplasm. Configurations with tubular elements were found in the cytoplasm of T. roseum, often in close association with the plasmalemma; and in vacuoles. These configurations appear to originate in the cytoplasm or between the plasmalemma and cell wall, and are later enclosed in the vacuoles, either in the living fungus or during preparation for electron microscopy.

Extracellular space in frozen and ethanol substituted central nervous tissue.

AbstractCerebellar and cerebral cortices were frozen under various conditions; within 30 seconds of circulatory arrest, after eight minutes of asphyxiation, after ten minutes of perfusion with glutaraldehyde and after perfusion with this fixative and osmium tetroxide postfixation. Ethanol was used as the solvent in freeze substitution of these tissues. The resulting EMs closely resembled those of similar material freeze substituted in acetone. There were no differences in extracellular space even though differences have been reported between the space in EMs of conventionally fixed material dehydrated with ethanol or acetone.

Variations in rabbit platelet morphology induced by different aldehyde prefixatives

<h2>Summary</h2> The fine structure of rabbit platelets prefixed in acetaldeyhde or paraformaldehyde is compared with that obtained after glutaraldehyde prefixation: osmium tetroxide post-fixation was used. Acetaldehyde prefixation causes platelets to swell, reduces the density of their matrix and causes a partial loss of the external platelet coat. Within the cytoplasm, very dense granules and microtubules are not seen; other organelles are preserved and the vacuolar system becomes very prominent. Platelets prefixed in paraformaldehyde are rounded. Their matrix is not very dense and their membranes are not well preserved. The number of very dense granules is reduced and microtubules are not seen. Other cytoplasmic organelles are preserved. Cytoplasmic microfibrillae may be seen after either acetaldehyde or paraformaldehyde prefixation but not after glutaraldehyde fixation. Apart from this, glutaraldehyde/osmium tetroxide fixation preserves platelet shape and ultra-structure best.

Ultrastructure of dyads in muscle fibers of Ascaris lumbricoides.

The dyads of Ascaris body muscle cells consist of flattened intracellular cisternae applied to the sarcolemma at the cell surface and along the length of T-tubules. In specimens prepared by conventional methods (glutaraldehyde fixation, osmium tetroxide postfixation, double staining of sections with uranyl acetate and lead hydroxide), both the sarcolemma and the limiting membrane of the cisterna exhibit unit membrane structure and the space between them is occupied by a layer of peg-shaped densities which is referred to as the subsarcolemmal lamina. The lumen of the cisterna contains a serrated layer of dense material referred to as the intracisternal lamina. In specimens fixed in glutaraldehyde, dehydrated, and then postfixed in phosphotungstic acid, with no exposure to osmium tetroxide or heavy metal stains, the membranous components of the dyads appear only as negative images, but the subsarcolemmal and intracisternal laminae still appear dense. Except for the lack of density in membranes and in glycogen deposits, the picture produced by the latter method is very much like that of tissue prepared by conventional methods.

Uptake of colloidal thorium dioxide by mast cells.

Mast cells from the peritoneal cavity of the rat were obtained at various times following in situ injection of a colloidal thorium dioxide preparation (Thorotrast). They were prepared for electron microscopy by aldehyde fixation, osmium tetroxide postfixation, and embedding in Epon. Thorotrast was rapidly taken up by mast cells through enhanced or newly elicited surface specializations. It was confined at first to large vesicles which moved to the Golgi area. Subsequently, in a matter of a few hours only, it became associated with progressively more mature granules, including "fully" mature ones. In addition to demonstrating a further phagocytic or pinocytotic activity of mast cells, the findings suggest that mast cell granules share a common membranous investment, and that substances from the tissue environment may theoretically percolate over and interact with the granules. Mast cell function could thus be served primarily by absorptive rather than secretory processes.

Changes in extracellular space of the mouse cerebral cortex during hydroxyadipaldehyde fixation and osmium tetroxide post-fixation.

ABSTRACT Perfusion of the cerebral cortex of mice with a 4·5 and 12·5 % hydroxyadipaldehyde (HAA) solution in a cacodylate buffer caused a biphasic change in the tissue conductivity. After a latency of a fraction of a minute the cortical conductivity dropped markedly, reaching a minimum in 1·5–2 min. Then the conductivity increased again. Electron micrographs (EMs) of material perfused with HAA for 1 5—20 min and post-fixed with osmium tetroxide showed electron-transparent swollen structures, some of which could be identified as dendritic. The extracellular space consisted of 100–200 Å slits between the tissue elements and larger spaces in bundles of small profiles (unmyelinated axons). Cortex frozen after 2 min perfusion with HAA and subjected to substitution in acetone containing 2 % OsO4 at—85 °C showed swollen (dendritic) structures and a paucity of extracellular material in accordance with the conductivity drop. Often tight junctions between the tissue elements were present. Tissue frozen after 15–20 min of HAA perfusion when the conductivity had increased again yielded EMs which were characterized by an abundance of extracellular space between the small profiles. The mitochondria in the swollen (dendritic) structures were enormously enlarged. Cortex perfused for 15–20 min with HAA, post-fixed with OsO4 and then freeze substituted produced EMs resembling those of tissue fixed in the same way but not subjected to freeze substitution. The examination of the fixation process by freeze substitution demonstrated a sequence of major changes in the fluid distribution of the tissue which precludes any direct relationship between the spaces in the normal and fixed tissue.

Anionic binding of ruthenium red in fish extraocular muscle.

Application of Luft's ruthenium red technique to extraocular muscle of Fundulus grandis reveals dense accumulations of ruthenium red-positive material along the sarcolemma, in the synaptic cleft of the myoneural junction and in the T tubules. The granular material in the terminal sacs of the sarcoplasmic reticulum (SR), which is consistently preserved by glutaraldehyde fixation and osmium tetroxide postfixation, also reacts with ruthenium red. The intracellular ruthenium red has been seen outside the SR only where the sarcolemma obviously has been disrupted.

Morphology of rigor--shortened bovine muscle and the effect of trypsin on pre- and postrigor myofibrils.

Bovine semitendinosus muscles were sampled immediately after death, after 24 hr postmortem with storage at 2 degrees , 16 degrees , or 37 degrees C, and after 312 hr postmortem with storage at 2 degrees and 16 degrees C. A biopsy technique was used to prevent shortening during glutaraldehyde fixation. Postfixation in osmium tetroxide was followed by embedding in an Epon-Araldite mixture. Bovine muscle was supercontracted after 24 hr storage at 27deg; but was only slightly contracted after storage at 16 degrees for 24 hr. Muscle held at 37 degrees for 24 hr was slightly less supercontracted than the 2 degrees muscle. Striking similarities existed between muscles stored at 16 degrees and at 2 degrees C for 312 hr. Both were slightly shortened with narrowed I bands and an area of increased density, probably due to overlap of thin filaments in the middle of the A band. Postmortem shortening was accompanied by banding-pattern changes similar to those predicted for contracting muscle by Huxley and Hanson's sliding filament model. Treatment of myofibrils with 0.05% trypsin resulted in a rapid loss of Z lines and, in supercontracted myofibrils, caused a return of the banding pattern of resting muscle.

The sarcoplasmic reticulum and its association with the T system in an insect.

The fine structure of the sarcoplasmic reticulum and the transverse tubular system of the femoral muscle of the cockroach, Leucophaea maderae, was studied after prefixation in glutaraldehyde, postfixation in osmium tetroxide, and embedding in Epon. The sarcoplasmic reticulum in this muscle reveals features not previously reported. The sarcoplasmic reticulum is abundant, consisting mainly of a fenestrated envelope which surrounds each myofibril at all levels in the sarcomere. This sarcoplasmic reticulum envelope is continuous transversally as well as longitudinally along the myofibrils. Dyadic junctions are formed by a single T system element which contacts the unfenestrated sarcoplasmic reticulum of adjacent myofibrils in an alternating manner at the ends of the A band. At the dyads, regularly spaced thickenings of the sarcoplasmic reticulum membranes bordering the dyadic spaces are noted. These thickenings, however, do not contact the T tubule membrane. Typical dyadic contacts also are seen between the cell surface membrane and sarcoplasmic reticulum. Z line-like material is seen in contact with the membranes of the cell surface and longitudinal branches of the T systems.

A MORPHOLOGIC AND CYTOCHEMICAL STUDY ON THE GREAT ALVEOLAR CELL

Lungs from marsupials, bats and rodents were studied by light and electron microscopy. In all three groups, the great alveolar cells exhibit similar morphologic and cytochemical characteristics. Cytoplasmic vacuoles seen in these cells by light microscopy correspond to cytosomes that are demonstrable in them by electron microscopy. Such cytosomes are osmiophilic, periodic acid-Schiff-positive and stainable with Sudan black after acetone extraction. After fixation in a mixture of aldehydes, followed by extraction in chloroform-methanol and postfixation in osmium tetroxide, cytosomes lose their osmiophilia. The cytoplasm of the great alveolar cell is notable for a loosely ordered granular endoplasmic reticulum, an extensive Golgi apparatus and numerous multivesicular bodies. Many forms transitional in appearance between multivesicular bodies and cytosomes are present. In these, osmiophilic matter occupies the intervesicular space. It is proposed that these bodies are the precursors of cytosomes. The cytosomes are interpreted to be products of the "lysosomal" system in this cell. Ultimately they are secreted onto the alveolar surface.

Ultrastructural organization of cilia and basal bodies of the epithelium of the choroid plexus in the chick embryo.

Ultrastructural studies were performed on normal and abnormal cilia and basal bodies associated with the choroidal epithelium of the chick embryo. Tissues were prepared in each of several fixatives including: 1% osmium tetroxide, in both phosphate and veronal acetate buffers; 2% glutaraldehyde, followed by postfixation in osmium tetroxide; 1% potassium permanganate in veronal acetate buffer. Normal cilia display the typical pattern of 9 peripheral doublets and 2 central fibers, as well as a system of 9 secondary fibers. The latter show distinct interconnections between peripheral and central fibers. Supernumerary fibers were found to occur in certain abnormal cilia. The basal body is complex, bearing 9 transitional fibers at the distal end and numerous cross-striated rootlets at the proximal end. The distal end of the basal body is delimited by a basal plate of moderate density. The tubular cylinder consists of 9 triple fibers. The C subfibers end at the basal plate, whereas subfibers A and B continue into the shaft of the cilium. The 9 transitional fibers radiate out from the distal end of the basal body, ending in bulblike terminal enlargements which are closely associated with the cell membrane in the area of the basal cup. One or 2 prominent basal feet project laterally from the basal body. These structures characteristically show several dense cross-bands and, on occasion, are found associated with microtubules.

The fine structure of Giardia muris.

Giardia is a noninvasive intestinal zooflagellate. This electron microscope study demonstrates the fine structure of the trophozoite of Giardia muris in the lumen of the duodenum of the mouse as it appears after combined glutaraldehyde and acrolein fixation and osmium tetroxide postfixation. Giardia muris is of teardrop shape, rounded anteriorly, with a convex dorsal surface and a concave ventral one. The anterior two-thirds of the ventral surface is modified to form an adhesive disc. The adhesive disc is divided into 2 lobes whose medial surfaces form the median groove. The marginal grooves are the spaces between the lateral crests of the adhesive disc and a protruding portion of the peripheral cytoplasm. The organism has 2 nuclei, 1 dorsal to each lobe of the adhesive disc. Between the anterior poles of the nuclei, basal bodies give rise to 8 paired flagella. The median body, unique to Giardia, is situated between the posterior poles of the nuclei. The cytoplasm contains 300-A granules that resemble particulate glycogen, 150- to 200-A granules that resemble ribosomes, and fusiform clefts. The dorsal portion of the cell periphery is occupied by a linear array of flattened vacuoles, some of which contain clusters of dense particles. The ventrolateral cytoplasm is composed of regularly packed coarse and fine filaments which extend as a striated flange around the adhesive disc. The adhesive disc is composed of a layer of microtubules which are joined to the cytoplasm by regularly spaced fibrous ribbons. The plasma membrane covers the ventral and lateral surfaces of the disc. The median body consists of an oval aggregate of curved microtubules. Microtubules extend ventrally from the median body to lie alongside the caudal flagella. The intracytoplasmic portions of the caudal, lateral, and anterior flagella course considerable distances, accompanied by hollow filaments adjacent to their outer doublets. The intracytoplasmic portions of the anterior flagella are accompanied also by finely granular rodlike bodies. No structures identifiable as mitochondria, smooth endoplasmic reticulum, the Golgi complex, lysosomes, or axostyles are recognized.

Electron microscopy of tick-borne fever agent in bovine and ovine phagocytizing leukocytes.

Tuomi, Jaakko (University of Helsinki, Helsinki, Finland), and C.-H. von Bonsdorff. Electron microscopy of tick-borne fever agent in bovine and ovine phagocytizing leukocytes. J. Bacteriol. 92:1478-1492. 1966.-Two strains of tick-borne fever agent, one more virulent to cattle and the other more virulent to sheep, were studied in thin sections of leukocyte concentrates of an affected calf and sheep, respectively. The calf was partially immune from previous infections. To obtain leukocyte concentrate, erythrocytes were lysed by ammonium chloride solution. Glutaraldehyde fixation and osmium tetroxide postfixation were used. Agent particles were detected in neutrophil and eosinophil granulocytes (occasionally also in monocytes) inside cytoplasmic vacuoles. The preparation from the sheep revealed that the tick-borne fever agent has a life cycle; it appeared as large particles, intermediate forms, and small particles. In the preparation from the calf, only large particles were observed, probably as a result of partial immunity. Large particles had a cell wall, plasma membrane, cytoplasm with ribosome-like granules, and nucleoid. They were often elongated, bearing closer resemblance to rickettsiae than to psittacosis-lymphogranuloma-trachoma (PLT) agents. The arrangement of large particles in vacuoles was variable and provided an explanation for the different forms of light microscopic tick-borne fever bodies. The intermediate forms and small particles resembled those of PLT organisms. Release of particles occurred by rupture of inclusions. In the calf also extrusion of whole inclusions was observed. This phenomenon was thought to reflect increased resistance of the cells. The large particle was considered to be the usual infective form of the agent. A middle-ground position of the agent between the genus Rickettsia and the PLT group is suggested.

THE ORGANIZATION OF CYTOPLASMIC COMPONENTS DURING THE PHASE OF CELL WALL THICKENING IN DIFFERENTIATING CAMBIAL DERIVATIVES OF ACER RUBRUM

Cambial derivatives of Acer rubrum have been examined at stages of their differentiation following fixation in 3% or 6% glutaraldehyde with a post fixation in osmium tetroxide. At early stages of development numerous free ribosomes are present in the cytoplasm, and elements of the endoplasmic reticulum tend to align themselves parallel to the cell surfaces. The plasma membrane is closely applied to the cell walls. During differentiation a complex system of cytoplasmic microtubules develops in the peripheral cytoplasm. These microtubules are oriented, mirroring the orientation of the most recently deposited microfibrils of the cell wall. The microtubules form a steep helix in the peripheral cytoplasm at the time of deposition of the middle layer of the secondary wall. During differentiation the free ribosomes disappear from the cytoplasm and numerous elements of rough endoplasmic reticulum with associated polyribosomes become more evident. In many cases the endoplasmic reticulum is associated with the cell surface. During the later stages of differentiation there are numerous inclusions between the cell wall and the plasma membrane.

Hydroxypropyl methacrylate, a new water-miscible embedding medium for electron microscopy.

Aldehyde-fixed rat tissues were variously dehydrated and impregnated in water-miscible 2-hydroxypropyl methacrylate (HPMA) containing 3 to 20 per cent water and 0.1 per cent alpha,alpha-azobisisobutyronitrile as catalyst for subsequent polymerization with ultraviolet light. Heat polymerization was also effective. Blocks of embedded tissue readily gave ultrathin sections, which required staining by uranyl acetate and/or lead stains to give adequate contrast for electron microscopy. The ultrastructure of pancreas, kidney, muscle, and intestine was well preserved by aldehyde fixation alone. Use of postfixation in osmium tetroxide or direct osmium tetroxide fixation was unsatisfactory. The fine structure of aldehyde-fixed liver from fasted rats was well preserved, whereas that from normal rats showed considerable disorganization and collapse, apparently because of extraction of glycogen during the embedding procedure. Enzymatic extraction of proteins by pepsin and of ribonucleic acid by ribonuclease after either formaldehyde or glutaraldehyde fixation was rapidly effected by direct treatment of ultrathin sections with solutions of the enzymes. In contrast, no digestion of chromatin by deoxyribonuclease could be detected. In spite of this present limitation, HPMA appears to have several advantages over earlier water-miscible embedding media for electron microscopy and to be particularly suitable for ultrastructural cytochemistry.

ELECTRON MICROSCOPIC OBSERVATIONS OF THE INFLUENCE OF DIFFERENT FIXATIVES ON THE APPEARANCE OF CELLULAR ULTRASTRUCTURE.

The cells of the proximal convoluted tubules of the rat kidney, mouse hepatic parenchymal cells, and the juxtaglomerular cells (“J. G. cells”) of the afferent arteriole in rabbit kidney served as test tissues for a study of the modifications of structure produced by 14 different fixation procedures using osmium tetroxide, a chrome-osmium solution, glutaraldehyde, and formaldehyde as fixatives. The fixative solutions containing various buffers were used either alone (osmium tetroxide-containing compounds) or in various combinations (aldehyde fixation with postfixation in osmium tetroxide). The most conspicuous differences in appearance following different fixation procedures were noted in the cytosomes of renal proximal tubular cells and the granules in the “J. G. cells”. However, virtually all cytoplasmic organelles showed some modification of structure with different fixatives. The evidence indicated that the chemical composition of the buffer used with OsO4 was an important factor which particularly affected the density of the cytosomal matrix in renal proximal tubular and “J. G. cells”. The density of the matrix of the cytosomes was, however, also influenced by the fixative itself as indicated by studies of aldehyde-fixed tissues. With these fixatives the ultrastructural appearance was not modified by the buffer. Clumping of nuclear chromatin along the nuclear membrane and around the nucleolus occurred following fixation in aldehyde and Dalton's chrome-osmium solution, whereas the chromatin was evenly distributed in the nucleus when buffered osmium tetroxide was utilized for fixation. A specific reaction of dichromate with the granule of the „J. G. cells“ seemed to take place rendering these granules electron opaque, thereby facilitating their identification in the light and electron microscope. The findings were discussed with particular attention to the interaction of fixatives and buffers with various tissue components.

ELECTRON MICROSCOPY STUDIES ON A SPONTANEOUS MASTOCYTOMA OF THE DOG

The ultrastructure of a dog mastocytoma examined with the electron microscope after fixation in glutaraldehyde, post-fixation in osmiumtetroxide and butyl-methyl methacrylate embedding is described. The ultrastructural characteristics with particular regard to the submicroscopic morphology of specific granules were studied in details, also with the aid of comparative observations on thick sections stained by Giemsa and toluidine blue. On the basis of their observations, the authors describe the following characteristics of neoplastic mastcells: microvilli, a well-developed Golgi region, centrioles, mithocondria, ribosomes, endoplasmic reticulum and 4 different types of granules. Other mastcells, with various degree of regressive phoenomena, had almost no microvilli, multiple interruptions of plasma membrane, mithocondrial swelling as well as vacuolar and fibrillar aspect of the cytoplasm. The morphology of different types of intracytoplasmic granules is discussed also in the light of parallel observations made by other authors. Expulsions of granules were not observed. The hypothesis of the phospholipidic nature of the lamellar component of granules is suggested.

Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation.

THE ALDEHYDES INTRODUCED IN THIS PAPER AND THE MORE APPROPRIATE CONCENTRATIONS FOR THEIR GENERAL USE AS FIXATIVES ARE: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4 degrees C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that-notable in the case of glutaraldehyde-was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.