Size changes of mouse ova during preparation for morphometric studies in the electron microscope

Summary The diameters of oocytes during successive steps of preparation for electron microscopy were measured in the light microscope. During glutaraldehyde fixation the diameter of the ova mainly reflected the osmolarity of the fixative vehicle. An increase in the diameter of the ova occurred with postfixation in OsO₄ depending on time of postfixation and also on osmolarity and OsO₄ concentration. During ethanol dehydration the cell diameter decreased to its lowest value and remained unchanged after embedment in Epon. A procedure that minimizes ova size changes during preparation for electron microscopy is elaborated.This investigation was supported by NIH PL 480 Research Agreement No. 05-030-1.     

Temporal bone morphology after systemic arterial perfusion or intralabyrinthine in situ immersion—I. Hair cells of the vestibular organs and the cochlea

The morphological preservation of hair cells of the inner ear was analysed following fixation with intra-arterial perfusion or local intralabyrinthine in situ immersion using six different fixatives of glutaraldehyde (441–876mosm/kg), three fixatives with combinations of glutaraldehyde/paraformaldehyde (917–1196mosm/kg) and 1% osmic tetroxide (346mosm/kg). The influence of added macromolecules such as dextran to the buffer solution was investigated with regard to fixation technique and type of fixative.The best results were obtained when using a direct (local) immersion of the labyrinth with 1% osmic tetroxide. Vascular perfusion is not recommended, independent of type of fixative used. Cochlear outer hair cells and vestibular hair cells type I are more vulnerable to alterations of osmolality and fixation technique than are inner hair cells and hair cells type II.     

MEASUREMENTS OF SOME CELLULAR CHANGES DURING THE FIXATION OF AMPHIBIAN ERYTHROCYTES WITH OSMIUM TETROXIDE SOLUTIONS

On average, 15 per cent of the total haemoglobin present in the blood of the newt Triturus cristatus was extracted during 45 minutes of fixation in Palade-Caulfield fixative. This extraction was reduced with fixatives buffered at pH 6.2 instead of pH 7.4. The addition of Ca⁺⁺ ions to a final concentration of 0.01 M in the fixative completely suppressed haemoglobin extraction. The effect of the pH, and the presence or absence of Ca⁺⁺ ions in the fixative, on the rate of haemoglobin extraction has been determined. During Palade-Caulfield fixation the average projected area of newt erythrocytes increased by 37 per cent, and after dehydration and embedding in Epon the average area was 25 per cent greater than that of the unfixed cell. Fixatives buffered at pH 6.2 and containing 0.01 M Ca⁺⁺ ions caused cellular shrinkage, with the average projected area decreasing by 10 per cent in the fixative. This shrinkage continued during dehydration, and the final average area of the erythrocytes in Epon was 26 per cent less than that of the unfixed cells. Similar measurements with erythrocytes of Amphiuma tridactylum showed that after Palade-Caulfield fixation the average cellular area was increased by 45 per cent, and after dehydration and embedding in Araldite it was 36 per cent greater than that of the unfixed cell. The average nuclear area increased by 35 per cent during fixation but after embedding it was 26 per cent greater than that of the unfixed nuclei. With a fixative at pH 6.2 containing 0.01 M Ca⁺⁺ ions, both the nucleus and the whole cell shrank during fixation. The nuclear area decreased by 20 per cent and the cellular area by 22 per cent. After dehydration and embedding in Araldite, the average nuclear area had decreased by 35 per cent and the cellular area by 40 per cent. It has been shown that OsO₄ fixation lowers the isoelectric points of haemoglobins and other proteins. This finding has been used in the interpretation of the observed cellular changes resulting from fixation.     

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.     

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.     

Kinetics of glutaraldehyde fixation of erythrocytes: size, deformability, form, osmotic and hemolytic properties.

Red blood cells interact with glutaraldehyde (GA) in a complex kinetic pattern of events. At a given GA concentration in phosphate buffered saline (PBS), the sequence of cell 'volume' response, as measured by resistive pulse spectroscopy (RPS), includes: an immediate response to the overall solution osmolality; a constant volume, latent phase; a rapid swelling phase; an intermediate constant volume phase; and a shrinkage phase to a final steady state volume. The final volume depends on fixative solution osmolality; for GA concentrations between 0.05% and 0.25% w/v, fixative osmolalities of less than 355 mosM, including 'isotonic', or greater than 355 mosM, lead to final cell volumes greater or less than native, respectively. Cell-membrane deformability decreases continuously and monotonically with time, as assessed by RPS. The rate of fixation is a direct function of GA concentration, in accordance with a derived empirical expression. The measured kinetic responses are related to considerations of cell size, deformability, and form, and to mechanisms involved in abrupt osmotic hemolysis.     

Parathyroid cell variants may be provoked during immersion fixation

Summary Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO₄ in the same buffers or, alternatively, in s-collidine.Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5–2% formaldehyde and 2.5–5% acrolein in 0.1 M Na-cacodylate with or without Ca²⁺ and Mg²⁺, Na-phosphate or Na/K-phosphate at 4°C followed by postfixation with 1% OsO₄ in the same buffers or in s-collidine containing sucrose, Ca²⁺ and Mg²⁺. This procedure largely abolished the occurence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Improved preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM)

Various fixation protocols were used in an attempt to improve preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM). Wash solutions and fixatives of different composition and osmolarity were tested. Paraformaldehyde and glutaraldehyde concentrations were varied between 0.5% and 3%. Ruthenium red was tested as an additive in both primary fixation and postfixation, or in postfixation alone. HR-LVSEM revealed various degrees of ruffing, folding, blebbing, and peeling off of the plasma membrane, as well as holes of different sizes. The plasma membrane overlying the acrosome and the connecting piece proved to be particularly sensitive to varying fixation conditions. Consistent topographical differences were revealed among the different domains over the sperm head. Most of the differences were considered to be artifacts. Their consistency, however, suggests that structural and biochemical differences exist either within the membrane or in the structures subjacent to the membrane. Primary fixation turned out to be less critical than postfixation. Preservation of a smooth plasma membrane without holes could only be achieved when primary fixation in low aldehyde concentrations, with or without ruthenium red, was followed by postfixation with OsO₄ and 1,000 ppm ruthenium red. Examination of thin sections of the same material confirmed that even a considerable number of small holes are difficult to detect in transmission electron microscopy. These results show that with the recent increase in resolution of LVSEM there is need for further effort to improve sample processing. © 1993 Wiley-Liss, Inc.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Summary Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO₄ after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO₄. Morphometric analysis revealed that microwave enhanced fixation led to larger mean cell volume, to larger cell surface area, and to larger suface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimiking exocytosis.     

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.     

Impact of glutaraldehyde versus glutaraldehyde-formaldehyde fixative on cell organization in fish corneal epithelium

The purpose of this study was to examine the impact of a low osmolality glutaraldehyde fixative and a high osmolality glutaraldehyde-formaldehyde fixative on the structural organization of a tissue that could be exposed to low and high osmolality environments. The corneas of freshwater trout were prepared for transmission and scanning electron microscopy using either a fixative of 2% glutaraldehyde in 60 mM cacodylate buffer (pH 7.8, 260 mOsm/l) or a fixative prepared by adding 2.5% glutaraldehyde to a solution of 1% formaldehyde and buffering the solution with 0.1 M cacodylate (pH 7.6, 850 mOsm/l; Karnovsky-type fixative). The corneal epithelial cell layer thickness was greater after glutaraldehyde compared to glutaraldehyde-formaldehyde fixation (67 vs 55 mum), as was the thickness of the superficial cells (5.1 vs 3.4 mum) and basal cells (43 vs 38 mum). The intermediate (wing) cells of the epithelium were, however, less thick after glutaraldehyde fixation (15 vs 18 mum). The width of the squamous, intermediate and basal cells was greater following glutaraldehyde fixation with the effect being greatest in the superficial layers and insignificant at the level of the basal cells. The results show that chemical fixatives with extremes of osmolality cannot only produce different cell sizes in a tissue but also determine the overall organization of the cells in a positional-dependent fashion.     

Evaluation of fixative composition,fixative storage,and fixation duration on the fine structure and volume of root-cell nucleoli

Summary— The effect of various combinations of three fixative compositions (glutaraldehyde buffered in veronal acetate, cacodylate, and piperazine-N, N'-bis[2-ethanesulfonic acid]—PIPES], two fixative storage times (fresh vs 6 weeks), and two fixation durations (3 h vs 9 days) on nucleolar fine structure and nucleolar volume in three root cell-types of oat seedlings (Avena sativa L, cv Seger) were evaluated. All fixatives show overall good preservation of fine structure. Nucleolar components are distinct and well delineated in cells fixed in solutions buffered with either cacodylate or veronal acetate; the components are more condensed when preserved in fixative buffered with PIPES. Nucleolar volume is greatest in cells fixed in the cacodylate fixative, and smallest in those preserved in the PIPES fixative. Among the treatments tested, the PIPES fixative evidently best maintains nucleolar volume. Distracting particulate deposits are abundant on nuclei and nucleoli in cells preserved in the veronal-acetate fixative. Contrary to common assumptions, aging of buffered fixative at room temperature for 6 weeks seems to affect neither the general quality of cellular preservation nor the pH of the fixatives, although nucleolar volume is reduced by such treatment. Long-period fixation (9 days) results in destruction of membrane integrity (mitochondria, plastids, ER), and shrinkage of organelles from the cytoplasm. Nucleolar volume is reduced with prolonged fixation.     

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.     

ALTERATION OF THE CONFORMATION OF PROTEINS IN RED BLOOD CELL MEMBRANES AND IN SOLUTION BY FIXATIVES USED IN ELECTRON MICROSCOPY

The effects of several commonly employed fixatives on the three-dimensional conformations of two soluble proteins and the protein of intact red blood cell membranes have been studied by means of circular dichroism measurements in the spectral region of the peptide absorption bands. The fixatives used produced significant and parallel conformational changes in all of the proteins, in the increasing order: glutaraldehyde; OsO₄; glutaraldehyde followed by OsO₄; and KMnO₄. The last two treatments obliterated most of the helical character of the proteins. The significance of these observations to the preparation of specimens for electron microscopy is discussed.     

STUDIES ON THE FIXATION OF ARTIFICIAL AND BACTERIAL DNA PLASMS FOR THE ELECTRON MICROSCOPY OF THIN SECTIONS

The process of fixation of DNA-containing plasms is investigated by macroscopical and electron microscopical observations on solutions of DNA, nucleohistones, as well as on bacterial nuclei. The following treatments were found to produce a gelation of a solution of DNA or nucleohistones: (a) OsO₄ fixation at pH 6 in the presence of amino acids (tryptone) and Ca⁺⁺. (b) Exposure to aqueous solutions of uranyl acetate. (c) Exposure to aqueous solutions of indium chloride. Observed in the electron microscope, these gels show a fine fibrillar material. From experiments in which solutions of DNA or nucleohistones are mixed with bacteria and treated together, it is concluded that the behavior of the bacterial nucleoplasm is similar to that of the DNA solutions. The appearance of birefringence indicates that uranyl acetate and indium chloride produce an orientation of the molecules of a DNA solution during gelation. Bacterial chromosomes fixed by these agents also show a certain order, while those fixed by the OsO₄-amino acid-Ca⁺⁺ formula do not. Whether or not the order can be considered to be artificial is discussed, and a tentative conclusion is presented: (a) Uranyl acetate may induce artificial order. (b) Fixatives which do not gel DNA probably result in the grossest artifacts. (c) OsO₄ fixation at pH 6 in the presence of amino acids (tryptone) and Ca⁺⁺ may give the most accurate preservation of the in vivo disposition of DNA (RK⁺ fixation).     

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     

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.     

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.     

Importance of the Fixative for Reliable Ultrastructural Preservation of Poikilohydric Plant Tissues. Observations on Dry, Partially, and Fully Hydrated Tissues ofSelaginella lepidophylla

Leaves of desiccated ‘resurrection plants’,Selaginellalepidophylla, were hydrated either through the roots of intactplants or as isolated organs. Air-dry tissue and samples at1, 4, 8 and 24 h (both detached and intact) of hydration wereprepared for electron microscopy using aldehyde fixatives ofdifferent osmotic strengths. Both dry and hydrated tissues werealso prepared using freeze substitution. Significant differencesin the ultrastructural preservation of these different sampleswere noted. There was a direct correlation between the osmolalityof both the fixative and the tissue with the quality of ultrastructuralpreservation. When the osmolality of the fixative was slightly(or even considerably) higher than that of the tissue, optimalpreservation was achieved. Freeze substitution, however, gavethe most faithful preservation of all subcellular compartments,despite the frequent presence of small ice crystals. Additionally,hydration of detached leaves for more than 4 h resulted in swellingdamage of the organelles and cytoplasm, regardless of the fixationprotocol. Broadly interpreted, the results of this study indicate thatan optimal preservation of plant cell and organelle ultrastructurecan be achieved by the use of high osmolality fixatives or,preferably, freeze substitution. These results are also importantin determining the method of hydration of poikilohydric samplesfor physiological studies and for interpretation of functionalchanges as related to the structural condition of the organelles.Copyright1997 Annals of Botany Company Selaginella; fixation; ultrastructure; dry; hydrated     

The potential of the immunogold-silver staining method for paraffin sections

Summary After perfusion fixation of the rat kidney with glutaraldehyde, and postfixation of the renal cortex with osmium-low ferrocyanide (40 mM OsO₄+6 mM K₄Fe(CN)₆ in 0.135 M phosphate buffer, pH 8.0), secondary lysosomes of proximal tubule cells carry acoat of electron dense material on the inner surface of the lysosomal membrane. This coat separates matrix and membrane of lysosomes, and corresponds in location and width to the electron translucent halo of conventionally processed lysosomes in TEM. The material which forms the coat, is stained by phosphotungstic acid at pH 0.3, and by periodic acid — thiocarbohydrazide — silver proteinate more intensively than the cell surface coat of the same cell; it contains a high concentration of hydroxyl,vicinal-glycol and α-aminoalcohol groups. Supported by the Deutsche Forschungsgemeinschaft (SFB 105)