Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds

Summary Several compounds of osmium^VIII, including potassium osmiamate and coordination complexes of OsO₄ with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO₄ in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation.The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO₄, has a negligible vapor pressure of OsO₄. It has the molecular formula C₆H₁₂N₄.2OsO₄ and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO₄) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO₄, and the odor of OsO₄ becomes apparent.Such solutions of osmeth are (0.25%) considerably less concentrated with respect to OsO₄ than solutions (1–2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO₄ appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO₄ solutions of equivalent concentrations. Our findings indicate that OsO₄ solutions of less than 1% may be satisfactorily utilized in many cytochemical studies.Osmeth is safer and more convenient to handle than OsO₄ because small amounts may be solubilized as needed. It should be considered as a substitute for OsO₄ in ultrastructural cytochemistry.This investigation was supported by NIH research grant number DE 02668 from the National Institute of Dental Research and by NIH grant number RR 05333 from the Division of Research Facilities and ResourcesVisiting Professor, Dental Research Center, University of North Carolina at Chapel Hill, Jan.-May, 1975. Supported in part by USPHS Grant HD 09209     

An Electron Microscope Study of Myelin Figures

In the electron microscope, thin sections of OsO₄-fixed myelin figures from the phospholipide fraction of human brain show a pattern of parallel dark lines with a repeating period of about 40 A. It is shown that the dark lines probably represent the reaction product of OsO₄ with double bonds in the fatty acid chains, thereby marking the central portion of one bimolecular lamella. The addition of globin results in dense lines 25 to 50 A wide that cover the surface of the myelin figures. When such a figure consists of only two bimolecular leaflets of lipide covered with globin, the structure shows striking similarity to the image of cell membranes in fixed tissue sections. A hypothetical schema is given of the molecular structure of the figure, and the distribution of OsO₄ in it.     

SILVER IMPREGNATION OF ULTRATHIN SECTIONS FOR ELECTRON MICROSCOPY

A new procedure is described for silver impregnation of thin sections for electron microscopy. Sections of various tissues, fixed in OsO₄ and embedded in methacrylate, were treated with an ammoniacal silver solution, directly or after oxidation with periodic acid or hydrogen peroxide. After OsO₄ fixation all cellular membranous systems exhibit a primary argentaffinity probably due to the reduction of ammoniacal silver solution by the reduced osmium bound to unsaturated lipids. Bleaching the sections with hydrogen peroxide removes the argentaffinity of protoplasmic structures. Treatment of the sections with periodic acid results in decreased argentaffinity of protoplasmic components while the argentaffinity of metaplasmic structures is greatly enhanced. The latter procedure appears particularly useful for enhancing the contrast of basement membranes.     

ELECTRON MICROSCOPE OBSERVATIONS ON FROZEN-DRIED CELLS

To explore the problem of artefacts which may be produced during usual fixation, dehydration, and embedding, the authors have examined pancreas, liver, and bone marrow frozen at about -180°C., dried, at -55 to -60°C., embedded in methacrylate, sectioned, and floated on a formol-alcohol mixture. By these treatments the labile structure of living cells can be fixed promptly and embedded in methacrylate avoiding possible artefacts caused by direct exposure to chemical fixatives. Cell structures are ultimately exposed to a fixative when the sections are floated on formol-alcohol, but at this stage artefacts due to chemical fixation are expected to be minimized, as the fixatives act on structures tightly packed in methacrylate polymer. In the central zone of tissue blocks so treated, the cells are severely damaged by ice crystallization but at the periphery of the blocks the cell structure is well preserved. In such peripherally located cells, elements of the endoplasmic reticulum (ER), Palade's granules, homogeneously dense mitochondria, and nuclear envelopes and pores, can be demonstrated without poststaining with OsO₄. The structural organization in the nucleus is distorted by vacuolization. The mitochondrial membranes and cristae, cellular membrane, and the Golgi apparatus, however, are detected only with difficulty. The Golgi region generally appears as a light zone, in which some ambiguous structures are encountered. After staining the sections with OsO₄ or Giemsa solution, an inner mitochondrial structure which resembles the cristae seen in conventional OsO₄-fixed specimens appears, but the limiting membrane is absent. Treatment with OsO₄ or Giemsa solution also renders more distinct the membrane of the ER and Palade's granules but not the Golgi apparatus and cellular membrane. Treatment with ribonuclease results in the disappearance of Palade's granules. On the strength of these observations the authors conclude that OsO₄ fixation gives a satisfactory preservation of such cell structures as the nuclear envelope, endoplasmic reticulum, and Palade's granules, though it may induce slight swelling of these cell components.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of Bombyx mori Linné were fixed in buffered (pH 8.2) 1% OsO₄ or 3 % KMnO₄ and thin sections of the tissue, embedded in methacrylate or epoxy Epon resin, were studied under the electron or light microscope.At the late stage of differentiation of the spermatid, the nucleus shows an elongated conical contour, being composed of fine fibrillar elements. These fibrillar elements fixed in OsO₄ measure 100 to 130 Å in diameter, while those fixed with KMnO₄ are approximately 70 Å in diameter.It has been found for the first time in the spermiogenesis of the silkworm that two bands and a tubular structure develop in close proximity to one another and attached to the plasma membrane of the spermatid. The two bands fixed in OsO₄ are electron dense, but in the material fixed with KMnO₄, one of them, situated within the cell body, is as dense as that fixed in OsO₄, while the other, outside the cell body, is much less dense. These apparently novel apparatuses develop from the caudal nuclear region along the elongating spermatid, but the dense band intertwines with the acrosome in the apical region of the nucleus along the major axis of spermatid, while the tubular structure and the clear band reach far into the nutritive cell where the dense band and acre-some are not visible.A possible relationship between the tubular structure and the nutritive cell has been discussed.This study was supported by Grant GM-8327-03 from the United States Public Health Service.     

Osmium tetroxide footprinting of a scaffold attachment region in the maizeAdh1 promoter

Osmium tetroxide (OsO₄) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO₄ hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO₄ will also identify other torsional perturbations of DNA. In this study, OsO₄ was used to footprint an AT-rich region (between –780 and –500) of the maizeAdh1 promoter. Hyperreactive sites were identified bothin vitro andin vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO₄ hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei.     

Formazan-Stabilität und -Reoxydation in Gewebsstrukturen unter dem Einfluß elektronenmikroskopischer Präparationstechnik

Summary The stability of NBT and TNBT towards embedding media used in electron microscopy (Methacrylat, Vestopal W, Araldit, Epon) and their ability to be re-oxidized was tested on formazan-containing tissue sections and trial stripes. The formazans of the monotetrazoles INT, MTT and TTC were included for comparative reasons. After having been exposed to osmium vapour or 2% OsO₄-solution over a period of 4 weeks formazan-containing trial stripes showed no changes whatsoever; the formazans of TTC, MTT, and INT were quantitatively soluble in ether after OsO₄ treatment, whereas the di-formazans of NBT and TNBT which are known to be stable to organic solvents, could not be extracted. The ether soluble formazans of TTC and MTT could be re-oxidized by OsO₄. INT-, MTTCo-, and NBT-formazan containing kidney sections showed no sign of re-oxidation after 1 hour fixation in 1% OsO₄-solution and storage for four years. It is therefore concluded that formazan which is bound to tissue lipoproteins is difficult to re-oxidize with OsO₄ in concentrations normally used in electron microscopy. Benzoyl peroxyd, which acts as an catalyser when methacrylat is used as embedding material, re-oxidized all formazans of the tetrazoles under test within a very short period of time. The components Activateur and Initiateur of the Vestopal W embedding substance also induced a (delayed) formazan re-oxidation; the formazans of NBT and TNBT showed no reaction to the components of Araldit and Epon embedding material, MNA excluded.The fixation of the tissues with various aldehydes (glutaraldehyde, hydroxyaldipaldehyde, crotonaldehyde, formaldehyde) followed by incubation in a TNBT-solution caused a temperature dependent reduction of the TNBT to brown di-formazan which may have some influence in the interpretation of enzyme-histochemical investigations.Zusammenfassung Es wurden NBT und TNBT hinsichtlich ihrer Stabilität gegenüber elektronenmikroskopischen Einbettungsmitteln (Methacrylat, Vestopal W, Araldit, Epon) sowie ihrer Reoxydationsfähigkeit (OsO₄, Benzoylperoxyd) an formazanhaltigen Gewebsschnitten und Teststreifen untersucht. Vergleichsweise kamen auch die Formazane der Monotetrazole INT, MTT sowie TTC zur Testung. — Formazanhaltige Teststreifen lassen nach 4wöchiger Einwirkung von Osmium-Dämpfen oder 2% iger OsO₄-Lösung keine Veränderungen erkennen; die Formazane vom TTC, MTT und INT lösen sich auch nach OsO₄-Behandlung quantitativ in Äther, während die in organischen Lösungsmitteln beständigen Diformazane vom NBT und TNBT sich nicht extrahieren lassen. Die in Äther gelösten Formazane vom TTC und MTT werden in kurzer Zeit durch OsO₄ reoxydiert. INT-, MTTCo- und NBT-formazanhaltige Nierenschnitte lassen nach Istündiger Fixierung in l% iger OsO₄-Lösung nach 4jähriger Aufbewahrung keine Reoxydationserseheinungen erkennen. Es wird daraus der Schluß gezogen, daß an Lipoproteinstrukturen des Gewebes gebundenes Formazan einer Reoxydation durch OsO₄ in den zur elektronenmikroskopischen Präparation verwendeten Konzentrationen schwer zugänglich ist. — Sämtliche Formazane der getesteten Tetrazole werden durch den bei der Methacrylat-Einbettung verwendeten Katalysator Benzoylperoxyd schon nach kurzer Einwirkungszeit vollständig zum Tetrazol reoxydiert. Durch die Komponenten Activateur und Initiateur der Vestopal-W-Einbettung erfolgt ebenfalls eine — wenn auch verzögerte — Formazan-Reoxydation, während die Formazane vom NBT und TNBT gegenüber den Komponenten der Araldit- und Epon-Einbettung (mit Ausnahme von MNA) keine Veränderungen erkennen lassen.Eine Vorfixierung von Gewebsproben in verschiedenen Aldehyden (Glutaraldehyd, Hydroxyadipaldehyd, Crotonaldehyd, Formaldehyd) und anschließende Inkubation in einer TNBT-Lösung bewirkt eine temperaturabhängige Reduktion des TNBT zum braunen Diformazan und dürfte daher von Einfluß auf die Interpretation enzymhistochemischer Untersuchungen sein.     

Fine Structure of Bacillus subtilis : I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO₄, OsO₄, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO₄ in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO₄ fixation appeared to provide much better definition of the boundaries of various structures than did OsO₄. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO₄ fixation) as two thin lines. In cells fixed first with OsO₄ solution, and then refixed with a mixture of KMnO₄ and OsO₄ solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO₄. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.     

SOME ELECTRON MICROSCOPICAL OBSERVATIONS ON LIQUID-CRYSTALLINE PHASES IN LIPID-WATER SYSTEMS

OsO₄ fixation preserves some liquid-crystalline phases of soaps and phospholipids to an extent that it is possible to observe their structure in electron micrographs of thin sections. Good agreement exists between the structure observed directly and that deduced from x-ray diffraction studies of the same systems.     

Relative speed of fixation of glutaraldehyde and osmic acid in plant cells measured by grana appearance in chloroplasts

Summary Brief fixation in a mixture of glutaraldehyde and OsO₄ caused stacked chloroplast grana membranes in leaf cells of wheat, barley, tobacco, maize, cowpea, pigweed or bean plants to distend and vesiculate. Fixation with glutaraldehyde followed by OsO₄ prevented this fixation artifact. In a fixative mixture, OsO₄ apparently reacted with cell contents before glutaraldehyde.     

Bacterial mesosomes: Real structures of artifacts?

The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO₄ fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO₄ in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO₄ in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO₄. Such damaging action was deduced from the observation that 0.1% OsO₄ quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K⁺ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO₄, 4% OsO₄ or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.     

THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO₄ with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO₄. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.     

INFLUENCE OF GLUTARALDEHYDE AND/OR OSMIUM TETROXIDE ON CELL VOLUME, ION CONTENT, MECHANICAL STABILITY, AND MEMBRANE PERMEABILITY OF EHRLICH ASCITES TUMOR CELLS

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO₄), and osmium tetroxide (OsO₄) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO₄ or OsO₄ but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO₄ but little or no effect with GA or GA-OsO₄. Osmolality of the buffer had a marked effect on cell volume with OsO₄, whereas with GA or GA-OsO₄ it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO₄ alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO₄. After fixation with GA and/or OsO₄ cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.     

Lichtmikroskopische Untersuchungen zur Strukturerhaltung farbstoffinduzierter autophagischer Vakuolen durch verschiedene Fixantien

Zusammenfassung Fibroblastenkulturen wurden mit Mepacrin (Atebrin®), Neutralrot und Toluidinblau unter vergleichbaren Bedingungen vitalgefärbt. Die Farbstoffe induzieren die Bildung autophagischer Vakuolen (Autolysosomen) im Cytoplasma. Die Eignung von sieben verschiedenen Fixantien zur Erhaltung dieser im lichtmikroskopischen Sinn neugebildeten Strukturen wurde untersucht.Kriterien der jeweiligen Fixationsleistung waren einmal die Erhaltung der autophagischen Vakuolen an sich, zum anderen die Erhaltung ihrer farbstoffabhängigen, morphologischen Individualität.Als wenig leistungsfähig haben sich erwiesen die Lösungen nach Carnoy und Bouin sowie Formol. Glutaraldehyd bewahrt die Lysosomenstruktur befriedigend, jedoch nicht ausreichend stabil für weitere, etwa histochemische, Eingriffe. Kaliumbichromat gewährleistet bessere Stabilität, jedoch nur geringe Lebensähnlichkeit der Autolysosomen.OsO₄ und NaMnO₄ sind den anderen Fixantien hinsichtlich der Erfüllung beider Kriterien deutlich überlegen. Die Befunde werden mit dem lipidstabilisierenden Effekt, den beide Metalloxydverbindungen an den phospholipidreichen Autolysosomen ausüben, in Zusammenhang gebracht.Unterschiede in der Wirkung ließen sich nach Anwendung von OsO₄ und NaMnO₄ an den AV nachweisen: Mepacrin-AV werden durch OsO₄ etwas lebensähnlicher erhalten als durch NaMnO₄. Die Neutralrot-AV und Toluidinblau-AV mit deutlicher vakuolärer Struktur werden nur durch Permanganat im Zusammenhang erhalten, mit deutlicher Abgrenzung der Toluidinblau-induzierten von den Neutralrot-induzierten Autolysosomen.Nach Osmium- und Permanganatfixation zeigen die Zellkulturen starke Affinität zu Methylenblau, nicht Eosin. Nur die OsO₄-fixierten Autolysosomen halten gegenüber Alkoholeinwirkung ihre Anfärbung im wesentlichen bei.Die Befunde werden diskutiert.

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Light microscopical investigations on structural preservation of Dye-Induced autophagic vacuoles by diverse fixatives

Summary Fibroblasts grown in monolayer were subjected to vital staining by mepacrine (Atebrine®), neutral red and toludine blue under comparable conditions. These dyes induce the formation of autophagic vacuoles (autolysosomes) in the cytoplasm. The preservation of these structures, which are considered to be newly formed in the dimension of the light microscope, by seven different fixatives has been examined.The criteria employed to assess the performance of each fixative consisted of 1. the preservation of the autophagic vacuoles per se and 2. their dye-dependent morphological characteristics.Fixation by Carnoy's or Bouin's solution as well as by formaline gave poor results. Glutaraldehyde preserved lysosomal structure satisfactorily, but not adequately for further application of histochemical methods. Potassium dichromate has a stabilizing effect on autophagic vacuoles, however, structures are not equivalent to those observed in living cells.Osmium tetroxide (OsO₄) and sodium permanganate (NaMnO₄) are superior to the other fixatives with regard to the afore mentioned criteria. These observations are explained by the wellknown lipid-stabilizing effect which both metal oxides are expected to exert on autolysosomes with their high content of phospholipids.After fixation with OsO₄ and NaMnO₄ diverging effects on autophagic vacuoles could be ascertained. Mepacrine-induced autophagic vacuoles are preserved somewhat more accurately by OsO₄ than by NaMnO₄.Autolysosomes induced by neutral red and toluidine blue display a more vacuolated appearance and are preserved as a whole only by permanganate. Distinct differences exist between autophagic vacuoles induced by toluidine blue and those induced by neutral red.After fixation by OsO₄ and NaMnO₄ cells from culture display a strong affinity to methylene blue, but not to eosin. The staining of autolysosomes by methylene blue is resistant to ethanol after fixation in OsO₄ only.The results are discussed.

Die Arbeit wurde mit Unterstützung der Firma Hoffmann-La Roche und Co. A.G., Basel, durchgeführt.     

ELECTRON MICROSCOPY OF SPORES OF BACILLUS MEGATERIUM WITH SPECIAL REFERENCE TO THE EFFECTS OF FIXATION AND THIN SECTIONING

Resting spores of Bacillus megaterium appear uniformly opaque and undifferentiated under the electron microscope. Germinated spores and spores which have lost their dipicolinic acid underwent characteristic changes in structure. Spores fixed with KMnO₄ lose their dipicolinic acid. Spores fixed with OsO₄ under certain conditions retain their dipicolinic acid. When conventional sectioning procedures are used with either method of fixation, abnormal spore structure is produced as a result of the solution of cellular constitutents. Dry sections of unfixed spores embedded in methacrylate reveal the spore structure in a more normal state. Indirect evidence has been obtained for the existence of a penetration barrier at or near the outer edge of the cortex.     

An electron microscope study of effects of various fixatives and thin-section enzyme treatments on a nuclear polyhedrosis virus

A comparison of fixatives and embedding media used in thin sectioning of polyhedra and isolated virions of the Pseudoplusia includens nuclear polyhedrosis virus demonstrated that best results are obtained with glutaraldehyde-OsO₄ fixation and epoxy embedding. Fixation was obtained with formaldehyde, acrolein-formaldehyde, or OsO₄ alone but the crystalline array of the polyhedral protein was not preserved. Glycol methacrylate embedding medium resulted in images of poor quality. Treatment of thin sections of polyhedra and virions with enzymes showed that the polyhedral membrane is resistant to digestion with proteases but the interiors of polyhedra were removed with pepsin, pronase, subtilisin, and a mixture of deoxyribonuclease and trypsin. Enzyme treatment of thin sections of virions resulted in removal of the nucleocapsid by all proteases tested except trypsin. A mixture of deoxyribonuclease and trypsin digested the nucleocapsid. The envelope of the virion resisted enzyme treatment.     

The Use of OsO4 Fixative for Feulgen-Stained Preparations

OsO₄ has many advantages over Carnoy's fixative mixture for the Feulgen nuclear staining in the protozoan Tokophrya infusionum. While Carnoy's fluid used prior to the Feulgen reaction produces shrinkage of the macronucleus and coarse clumping of its chromatin bodies, OsO₄ preserves faithfully the size and shape of the macronucleus and its chromatin material. This finding seems to be of special importance in view of the fact that electron microscopy relies on OsO₄ fixation. The satisfactory preservation of structured detail in Feulgen-stained preparations is of importance for the correlation of histochemical and morphological information.     

Osmium tetroxide as a histochemical and histological reagent

Summary From the evidence discussed it can be concluded that osmium tetroxide (OsO₄) would be reduced to black OsO₂ (or an equivalent compound) by the ethylene bonds of liquid or solid cis-unsaturated lipids or by the ⁵-double bond in cholesterol in solid state in tissues. No evidence has been obtained to suggest that OsO₄ is either reduced or bound by proteins and polysaccharides in tissue-sections.     

Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy

Summary Specimens infused with or suspended in a mixture of 10–30% poly(vinylpyrrolidone) and 2.07–1.61m sucrose can often be more easily frozen-sectioned than those infused with sucrose alone. The pH of such a mixture can be efficiently adjusted to neutrality by using Na₂CO₃. Use of poly(vinylpyrrolidone) causes little or no increase in the background level of immunolabelling. Adsorption staining of ultrathin frozen sections with a mixture of uranyl acetate and poly(vinyl alcohol), i.e. a simple thin-embedding of the sections in such a mixture, produces positive staining effects that are often enough to delineate structures of many organelles. When OsO₄-treated frozen sections are stained with uranyl acetate and further adsorption-stained with a mixture of lead citrate and poly(vinyl alcohol), the overall staining effects are similar to those observed in double-stained conventional sections.A large portion of the findings was reported as a part of the author's presentation in the 11th International Congress on Electron Microscopy, held in Kyoto, Japan, in 1986.     

Silicotungstic acid for cytochemical localization of water soluble substance(s) of cholinergic motor nerve terminal

Summary Silicotungstic acid (STA), an electron dense substance and a powerful precipitating agent of quaternary ammonium salts such as choline and acetylcholine, was employed on the frog motor end-plate in order to prove that STA reacts with diffusible substance(s) in nerve terminals. Thus, STA treatment and osmium tetroxide (OsO₄) fixation were performed in three different ways. No reaction was detectable when STA treatment followed osmification, while simultaneous treatment with STA and OsO₄ darkened both presynaptic and synaptic vesicle membranes. When STA was employed directly on fresh tissues which were subsequently fixed by OsO₄, small black precipitates were observed in the synaptic vesicles and none on other synaptic structures. The possible reaction of STA with acetylcholine is discussed.