Vapor Fixation of Intractable Fungal Cells for Simple and Versatile Scanning Electron Microscopy

Imaging of exudation of conidia from fruiting bodies by scanning electron microscopy is challenging because of the difficulty in achieving specimen fixation that enables conidia to be immobilized from fruiting bodies. Conidia in chains borne on conidiophores are readily dissipated on coming in contact with liquid fixatives before fixation becomes effective. Two phialide‐forming fungi including Aspergillus and Penicillium species, and a pycnidium‐forming fungus Fusicoccum species were subjected to a vapor fixation procedure for scanning electron microscopy rather than conventional immersion fixation procedures. The intractable fungi were exposed to the vapor of 2% glutaraldehyde and 2% osmium tetroxide each for at least 2 h, subsequently followed by gold coating. Through the vapor fixation, excellent retention of conidial chains in phialides was achieved in this study. It appeared that conidial masses from pycnidia were usually held together in globose to oval drops. The simple vapor fixation procedure for preserving delicate fungal cells could be widely used for routine applications to facilitate morphological characterization of diverse plant pathogenic fungi as they are in ex planta ecological niches during the saprophytic phase as well as in host parts during the parasitic phase.     

The ultrastructure of Candida krusei

Various methods of chemical fixation and freeze-drying of Candida krusei were compared to determine the most appropriate method for the ultrastructural investigation of the thick walled organisms of this genus. Freeze-drying without chemical fixation was of little value because of insufficient variation in electron density. Potassium permanganate was able to penetrate the intact cell but failed to show cytoplasmic glycogen and lipid and some details of the cell wall. While normal glutaraldehyde, formaldehyde and osmium tetroxide treatment failed to permeate and preserve intracellular structures, several cycles of rapid freezing (–155°C) and thawing followed by glutaraldehyde fixation and osmium tetroxide post-fixation demonstrated the intracellular details of the majority of cells so treated.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

Swelling of golgi apparatus cisternae in monensin-treated tissues is modulated by fixation conditions

Summary Swelling of Golgi apparatus cisternae is reported to be a common response to the ionophore, monensin. However, the amount of swelling depends on fixation, thus raising the question of whether the swelling response is due to monensin or to the fixation protocol. To resolve this problem, maize root cap cells were treated with monensin and then fixed with glutaraldehyde and osmium tetroxide (applied sequentially), osmium tetroxide alone, or aqueous potassium permanganate, or were quick frozen in liquid propane and substituted in acetone-osmium tetroxide. The chemical fixatives (which take minutes to stabilize tissue elements) were judged by comparison with freeze substitution which requires only fractions of a second to stabilize tissue elements. The results verify that monensin causes cisternal swelling and that this swelling is best observed at the ultrastructural level by fixation in glutaraldehyde/osmium tetroxide or by freeze substitution.     

Exocytotic release of secretory granules from endocrine cells in the midgut of insects

Summary Exocytotic release of the secretory granules of the endocrine cells in the midgut of a cockroach, Periplaneta americana, was studied by means of fixation with tannic acid in combination with glutaraldehyde and osmium tetroxide. A sequence of images indicative of exocytosis suggests the following steps in this process: (1) A delicate connection appears between the granule-limiting membrane and the plasma membrane. (2) The plasma membrane approaches the granule, forming a concave indentation. (3) The granule-limiting membrane fuses with the plasma membrane and opens to give rise to an omega profile. (4) The granule content is voided into extracellular space. Exocytosis occurs not only at the base of the cell but occasionally at its side facing adjacent cells. (5) The exocytotic invagination after release becomes smaller and narrower; sometimes a coated pit with bristles appears. Multiple exocytosis, and exocytosis in the endocrine cells of the nidus, i.e., the regenerative cell mass, are also described.     

The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate

Synopsis A densitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.     

Fluidity of phospholipid bilayers and membranes after exposure to osmium tetroxide and gluteraldehyde

The response of fluid bilayer regions to osmium tetroxide and glutaraldehyde fixation was examined in phospholipid multilayers and in nerve bundles from the walking legs of the lobster Homarus americanus. The samples were spinlabeled either with 5-doxylstearic acid (the 4′4′-dimethyloxazolidine-N-ozyl derivative of 5-ketostearic acid) or the maleimide spin label, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl. Osmium tetroxide fixation abolishes the characteristic orientation of the spin-labeled lipid bilayer regions and virtually eliminates motion on the electron spin resonance time scale. Glutaraldehyde treatment reduces the motion of maleimide spin labels covalently attached to proteins. However, in contrast to osmium tetroxide fixation, glutaraldehyde has essentially no effect on the orientation and mobility in the fluid bilayer regions, and hence probably does not restrict directly the potential for translational motion in membrane phospholipid bilayer regions.     

ULTRASTRUCTURAL LOCALIZATION OF DIALYZED IRON-REACTIVE MUCOSUBSTANCE IN RABBIT HETEROPHILS, BASOPHILS, AND EOSINOPHILS

For ultrastructural localization of acid mucosubstances in rabbit granulocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde, or osmium tetroxide, sectioned at 40 µ, and stained with the Rinehart and Abul-Haj solution of dialyzed iron (DI). Heterophils revealed DI staining on the outer surface of the plasma membrane, in the Golgi complex involved in primary granulogenesis, and in primary granules. The intragranular distribution of DI-stained material varied at different stages in the maturation of primary granules. Immature granules of heterophils fixed by any of the three methods contained a peripheral concentric band of DI-positive material; however, fully mature primary granules possessed a core of DI-reactive material in heterophils fixed with osmium tetroxide, but they contained little or no staining in heterophils fixed with formalin or glutaraldehyde. Secondary granules of rabbit heterophils failed to stain with DI. Tertiary granules, observed only in late heterophils, contained distinct DI-positive particles. Basophil granules exhibited intensely DI-stained material distributed in an orderly pattern throughout the granule. In eosinophils, DI staining was localized in the Golgi complex and in the rims of a few immature cytoplasmic granules.     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.     

On the stabilization by fixation of configurational states in beef heart mitochondria

The energized configuration of the cristal membrane of beef heart mitochondria can be maintained only as long as oxygen is available for electron transfer. When the oxygen supply is exhausted, the membrane undergoes a transition to the nonenergized configuration. Since the exhaustion of the available oxygen supply is complete in 5–20 sec, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is the most effective way of freezing the configurational state of the cristal membrane. Fixation with glutaraldehyde appears to be complete within 1–2 sec even at 0°. Osmium tetroxide alone can also freeze the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). The freezing by fixation of the cristal membrane in the aggregated configuration is best accomplished by the sequential use of glutaraldehyde and osmium tetroxide. When the levels of glutaraldehyde and osmium tetroxide are respectively too low or too high, the mitochondrion will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering studies provide an independent method for monitoring configurational changes in mitochondria; these light-scattering measurements confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration as judged by absence of light-scattering changes after the fixatives are introduced. Reagents used in negative staining will induce the geometrical form of the energized configuration of the mitochondrion even under nonenergizing conditions. These reagents are thus unsuitable for use in studies of configurational transitions in mitochondria.     

Ultrastructural localization of calcium in prothoracic gland cells of Galleria mellonella L. (Insecta,Lepidoptera) in relation to secretory activity

Summary Localization of intracellular calcium was demonstrated by precipitation with potassium hexahydroxoantimonate in the fixation medium containing osmium tetroxide or osmium tetroxide and glutaraldehyde. The presence of calcium in the precipitates was confirmed by X-ray microanalysis. Cells from active prothoracic glands contain more calcium deposits than inactive glands. The calcium precipitates are mainly localized in the nucleus, in the smooth endoplasmic reticulum, in the hyaloplasm and to a lesser degree in the mitochondria. These findings are consistent with the proposed role of calcium in the stimulation of steroidogenesis.     

Cellular membrane contrast and contrast differentiation with osmium triazole and tetrazole complexes

Summary Addition of heterocyclic nitrogen compounds to the classical osmium tetroxide postfixation medium, applied after glutaraldehyde fixation, results in enhanced membrane contrast in ultrathin sections of liver tissue. The addition of similar compounds to potassium osmate solutions, results in contrast differences in some cellular membranes. The membranes of the rough endoplasmic reticulum, the nuclear envelope and the plasma membrane acquire contrast, while the mitochondrial membranes do not. The apolar regions of membranes are contrasted when osmium tetroxide is combined with heterocyclic nitrogen compounds, whereas the polar regions are contrasted by combinations of potassium osmate with these compounds. This polar membrane contrast is probably due to the presence of an amino-group in the heterocyclic nitrogen compounds. Compounds without the amino-group do not contrast membranes, although the glycogen is contrasted.X-ray microanalysis served to establish the relative osmium content in contrasted glycogen, and showed that such nitrogen compounds play a role in complexation of cations in aldehyde-fixed tissues. Electron spectroscopy for chemical analysis (ESCA) measurements of isolated muscle glycogen show that after treatment with various osmium tetroxide or potassium osmate solutions, hexavalent and quadrivalent osmium species are present in the glycogen. The presence of (heterocyclic) nitrogen compounds in such solutions stabilizes certain osmium valency species, and this may account for the contrast observed.     

The release of membrane-associated calcium from rabbit neutrophils by fixatives. Implications for the use of antimonate staining to localize calcium

Summary The addition of oxalate to a suspension of rabbit peritoneal neutrophils before fixation with glutaraldehyde and postfixation with osmium tetroxide-antimonate greatly enhanced the amount of calcium antimonate precipitate subsequently detectable with the electron microscope. Using chlortetracycline as a fluorescent probe for membrane-associated calcium, it was found that both glutaraldehyde and osmium tetroxide release calcium from membrane-associated stores in suspensions of living neutrophils. These findings suggest that some of the calcium released from cellular stores during fixation with glutaraldehyde is trapped within the neutrophil by oxalate which then reacts with potassium antimonate. This produces a more copious precipitate of calcium antimonate than fixation without oxalate. It is suggested, therefore, that the histochemical localization of calcium by antimonate techniques may not always represent thein vivo situation. The use of oxalate during fixation, however, may give a better indication of the amount of calcium stored within a cell.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

THE EFFECTS OF RESERPINE AND HYPOXIA ON THE AMINE-STORING GRANULES OF THE HAMSTER CAROTID BODY

The carotid bodies from control, reserpine-treated, and hypoxia-treated hamsters were fixed with phosphate-buffered glutaraldehyde and osmium tetroxide, s-Collidine-buffered osmium tetroxide, or phosphate-buffered glutaraldehyde followed by potassium dichromate incubation. Following glutaraldehyde-osmium tetroxide fixation no differences in density or population of the electron-opaque granules in the glomus cells of either control or experimental animals were observed. With s-Collidine-buffered osmium tetroxide and the glutaraldehyde-dichromate technique a marked decrease in density without an appreciable reduction in number of granules was noted after reserpine treatment, while in hypoxia-treated hamsters the density and population of the granules were not different from those of the controls. The results indicate that reserpine depletes the amines without granule disappearance and that hypoxia does not affect the amine content of the granules. It is suggested that following glutaraldehyde-osmium tetroxide double fixation, persistence of the density of the granules in reserpine-treated animals is due primarily to the nonamine content, and that the amines in the glomus cells are probably not directly involved in the respiratory reflex.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds

Summary Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study, I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32–34 s, final temperature between 40° C and 47° C.     

Osmolarity of osmium tetroxide and glutaraldehyde fixatives

Synopsis The evidence available to date for the importance of fixative osmolarity is considered together with some observations on the volume changes of crab axons after fixation by osmium tetroxide and glutaraldehyde. The results obtained are compared with those obtained from crab axons and from amphioxus skin cells which had been processed and examined with the electron microscope after initial fixation in fixatives of different composition. It is concluded that the osmolarity of the fixative vehicle is of considerable importance when the fixing agent is glutaraldehyde but is of less importance when the fixing agent is osmium tetroxide or a mixture of the two agents.Preliminary observations upon crab axons fixed with glutaraldehyde in a vehicle approximating to the internal composition of the cells suggest that this approach to the design of fixative vehicles may be useful.     

Immersion and perfusion fixed rat adrenal medulla: The problem of mixed cells,clear cells,and the mode of secretion

Summary The adrenal medulla of the rat was studied utilizing various methods of fixation. In adrenal medulla specimens after immersion fixation either with glutaraldehyde or osmium tetroxide, elements such as mixed, clear, syncytial, or plasmodial cells, believed to be of artifactual origin, are observed in all of this material examined. These elements are absent in the specimens prepared by perfusion fixation. In specimens prepared by immersion fixation, secretory granules are found in close proximity to the plasma membrane; this localization is infrequent after perfusion fixation.Current theories of the mechanism of secretion of adrenal medullary hormones are discussed on the basis of our results. This investigation demonstrates the advantage and necessity of perfusion fixation in the study of the adrenal medulla.Supported by the Deutsche Forschungsgemeinschaft, grant No. Fo 77/1.     

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.     

The effects of 6-hydroxydopamine on the appearance of granulated vesicles in glomus cells of the rat carotid body

The glomus cells of the rat carotid body reveal an intense fluorescence after exposure to paraformaldehyde vapor and contain catecholamines. After initial fixation in glutaraldehyde, many granulated vesicles are seen in the glomus cells. After initial fixation in osmium tetroxide, most of the vesicles are depleted of their dense interiors and granulated vesicles occur infrequently. Administration of 6-hydroxydopamine followed by initial fixation in osmium tetroxide leads to the reappearance of dense interiors in virtually all vesicles. 6-Hydroxydopamine apparently is taken up by the membrane pump of the glomus cell and is incorporated into the amine storage granules, thereby displacing the endogenous monoamines. Osmium tetroxide does not dissolve the 6-hydroxydopamine from the vesicles, as it apparently does for the normal vesicular contents. The 6-hydroxydopamine does not fluoresce, hence 6-hydroxydopamine administration results in a decreased intensity of formaldehyde induced fluorescence in the glomus cells. Administration of reserpine after 6-hydroxydopamine treatment (and subsequent initial fixation in osmium tetroxide) depletes the previously restored dense material from the vesicles of the glomus cells. 6-Hydroxydopamine acts like a monoamine in that it is taken up by the glomus cell, incorporated into the vesicles, and can be depleted from the vesicles by reserpine.