The fine structure of blood cells in the ascidian Perophora viridis

The fine structure of each of the blood cell types of Perophora viridis has been characterized and strong evidence for localization of vanadium in two of these types is given. There are eight cell types; phagocytes which may contain completely engulfed cells, lymphocytes with a prominant nucleolus and scanty cytoplasm packed with clustered ribosomes, and six other cell types each with distinctive granules. Morula cells contain a central nucleus and cytoplasm filled by wedged bodies, about five of which are seen in section. These bodies contain regularly spaced electron dense foci. Green cells have the same organization but contain bodies which are electron dense throughout. Granular amoebocytes contain many smaller lightly staining oval bodies and much glycogen. Another cell type (probably orange cells of light microscopy) contains numerous granular rounded bodies. Compartment cells have vacuoles containing electron dense particles and signet ring cells have usually one large vacuole which is electron dense lined and may contain electron dense particles. Developmental stages of these cell types show involvement of endoplasmic reticulum and Golgi bodies in granule formation. After glutaraldehyde fixation alone the only extremely electron dense components are particles in the compartment cells and signet ring cells implicating these as sites of vanadium localization, although not excluding other cell types.     

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.     

Confocal scanning light microscopy of the Escherichia coli nucleoid: comparison with phase-contrast and electron microscope images.

The nucleoid of living and OsO4- or glutaraldehyde-fixed cells of Escherichia coli strains was studied with a phase-contrast microscope, a confocal scanning light microscope, and an electron microscope. The trustworthiness of the images obtained with the confocal scanning light microscope was investigated by comparison with phase-contrast micrographs and reconstructions based on serially sectioned material of DNA-containing and DNA-less cells. This comparison showed higher resolution of the confocal scanning light microscope as compared with the phase-contrast microscope, and agreement with results obtained with the electron microscope. The effects of fixation on the structure of the nucleoid were studied in E. coli B/r H266. Confocal scanning light micrographs and electron microscopic reconstructions showed that the shape of the nucleoid remained similar after OsO4 or glutaraldehyde fixation; however, the OsO4 nucleoid appeared to be somewhat smaller and more centralized within the cell.     

Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid

After standard glutaraldehyde-osmium tetroxide fixation procedures, the majority of microfilament bundles in BHK-21 cells exhibit relatively uniform electron density along their long axes. The inclusion of tannic acid in the glutaraldehyde fixation solution results in obvious electron density shifts along the majority of microfilament bundles. Striated patterens are frequently observed which consist of regularly spaced electron dense (D) and electron lucid (L) bands. A striated pattern is also observed along many BHK-21 stress fibers after processing for indirect immunofluorescence utilizing BHK-21 myosin antiserum. A direct correlation of these periodicities seen by light and electron microscope techniques is impossible at the present time. However, comparative measurements indicate that the overall patterns seen in the immunofluorescence and electron microscope preparations are similar. The ultrastructural results provide an initial clue for the ultimate determination of the supramolecular organization of contracile proteins other than actin within the microfilament bundles of non-muscle cells.     

PHYSICOCHEMICAL EFFECTS OF ALDEHYDES ON THE HUMAN ERYTHROCYTE

The effects of formaldehyde, acetaldehyde, and glutaraldehyde on human red blood cells were investigated. It was found that (a) The surface negative charge of the erythrocytes at pH 7 was increased 10% by glutaraldehyde, but not by the other two aldehydes. (b) The effect of incomplete fixation of the red blood cells was demonstrated by hemoglobin leakage studies The leakage of hemoglobin subsequent to formaldehyde treatment was especially pronounced Acetaldehyde-fixed cells showed some leakage of hemoglobin after an hour of exposure to the fixative, whereas glutaraldehyde-fixed cells showed no hemoglobin leakage. (c) All three aldehydes caused K⁺ leakage during fixation. The concentrations of K⁺ in the fixing solutions all reached the same level, but whereas the leakage with glutaraldehyde was immediate, that with formaldehyde was more gradual and that with acetaldehyde reached a steady state only after 24 hr. (d) The effects of the aldehydes on red cell deformability and swelling revealed that glutaraldehyde hardened the cells within 15 min, formaldehyde within 5 hr, while acetaldehyde required at least 24 hr to produce appreciable fixation. (e) The hematocrit changes accompanying the fixation process depended upon cell volume changes and loss of deformability.     

Comparative ultrastructure of a mucoid strain of Pseudomonas aeruginosa isolated from cystic fibrosis patient and its spontaneous non-mucoid mutant

The ultrastructure of a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin and its spontaneous non-mucoid variant was compared by transmission electron microscopy. Negatively-stained preparations of the mucoid strain obtained from plate cultures demonstrated dense, fibrous material projecting from the cell. No such material was observed in thin-sections or in negatively-strained preparations from liquid cultures. Thin-sections of ethanol-precipitated extracellular material from liquid cultures of the mucoid-strain revealed a cottony mesh of thin electron dense fibres. The non-mucoid strain did not produce such material. When prefixed with glutaraldehyde/malachite green mixture, cells of both strains demonstrated electron dense intracellular and extracellular malachite green-stainable structures. The internal complexes were frequently associated with the nucleoid or cell membrane and were replaced by electron transparent areas in cells prefixed with glutaraldehyde alone. Aeruginocins of the R-type were observed in mitomycin C induced cultures of both strains. Bacteriophages with 'claw-shaped' tail-tips were observed in the mucoid strain. Crystalline material was produced by the mucoid strain but only when plated on certain media.     

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.     

Improved preservation of alkaline phosphatase in salivary glands of the cat

Synopsis The effects of tissue preparation on the localization of alkaline phosphatase were assessed at the light and electron microscopical levels. Glutaraldehyde-containing solutions were more inhibitory than formaldehyde alone but appeared to give betterin situ immobilization of the enzyme. The inhibitory effects of glutaraldehyde solutions were related especially to temperature and time and not necessarily to material absorbing at 235 nm. Distilled glutaraldehyde was the only form of glutaraldehyde to give consistently good results with least inhibition. Snap-freezing of pre-fixed tissue, after washing in a sucrose-containing solution, gave satisfactory results without undue ice-crystal formation. Immersion in dimethylsulphoxide before snap-freezing gave less good localization with greater diffusion of reaction product. Use of a Sorvall tissue-chopper on unfrozen tissue did not produce satisfactory sections. Free-floating sections of pre-fixed material appeared less inhibited than glass-mounted sections. The most satisfactory results were obtained after per-arterial perfusion with a 1% distilled glutaraldehyde-0.8% formaldehyde mixture, containing dextran, for up to 5 min followed by formaldehyde-dextran. The results were uniform; there was stronger staining of stromal surfaces of myoepithelial cells than previously, basal duct cells were also stained and occasional staining between acinar cells was now evident for the first time.     

Fixation with even small quantities of glutaraldehyde affects red blood cell surface properties in a cell- and species-dependent manner. Studies by cell partitioning

Partitioning in charge-sensitive dextran-poly(ethylene glycol) aqueous phase systems reveals that fixation with even small concentrations of glutaraldehyde (e.g., 0.1% w/v) changes the surface properties of cells. While fixation with larger concentrations of glutaraldehyde (i.e., 1.85%) increases erythrocyte partition ratios, the effect of lower glutaraldehyde concentrations on the partition ratios appears to be species-specific. The differential effect of glutaraldehyde on rat reticulocytes and erythrocytes indicates that fixation is also cell-dependent. These data, together with the previous report that glutaraldehyde fixation does not change the characteristicrelative partition ratios of rat mature erythrocytes of different cell ages, suggest that the nature and extent of glutaraldehyde alteration of cell surfaces must, in each case, be empirically evaluated.     

A FIBER APPARATUS IN THE NUCLEUS OF THE YEAST CELL

The structure and mode of division of the nucleus of budding yeast cells have been studied by phase-contrast microscopy during life and by ordinary microscopy after Helly fixation. The components of the nucleus were differentially stained by the Feulgen procedure, with Giemsa solution after hydrolysis, and with iron alum haematoxylin. New information was obtained in cells fixed in Helly's by directly staining them with 0.005% acid fuchsin in 1% acetic acid in water. Electron micrographs have been made of sections of cells that were first fixed with 3% glutaraldehyde, then divested of their walls with snail juice, and postfixed with osmium tetroxide. Light and electron microscopy have given concordant information about the organization of the yeast nucleus. A peripheral segment of the nucleus is occupied by relatively dense matter (the "peripheral cluster" of Mundkur) which is Feulgen negative. The greater part of the nucleus is filled with fine-grained Feulgen-positive matter of low density in which chromosomes could not be identified. Chromosomes become visible in this region under the light microscope at meiosis. In the chromatin lies a short fiber with strong affinity for acid fuchsin. The nucleus divides by elongation and constriction, and during this process the fiber becomes long and thin. Electron microscopy has resolved it into a bundle of dark-edged 150 to 180 A filaments which extends between "centriolar plaques" that are attached to the nuclear envelope.     

Cyto- and histochemical demonstration of lignins in plant cell walls: an evaluation of the chlorine water/ethanolamine-silver nitrate method of Coppick and Fowler

Summary The chlorine water/ethanolamine-silver nitrate method introduced by Coppick and Fowler for the detection of lignins was evaluated for cyto- and histochemical work using different reagents and fixatives for specimens embedded in epoxy resin. Fixation schedules tested included ethanol, glutaraldehyde, and glutaraldehyde followed by O_sO₄ as a post-fixative. Chlorine water, sodium hypochlorite, and calcium hypochlorite were the oxidising agents evaluated for their efficacy as part of the Coppick and Fowler procedure. The Coppick and Fowler method was tested against stem woody tissue ofLophomyrtus obcordata, and haustorial xylem tissue of the sucker of its attached dwarf mistletoeKorthalsella lindsayi. The presence of lignins in walls of these cells was indicated in thin sections for transmission electron microscopy by fine electron-dense deposits. Post-staining thin sections did not affect the lignin reaction, but tended to mask its effect due to increased wall contrast. In histological preparations lignified walls stained orange/brown. Counter-staining in methylene blue/azur B caused lignified walls to appear dark green/brown and non-lignified walls blue. Fixation in either ethanol or glutaraldehyde produced identical staining for lignins. Penetration by chlorine water was sometimes irregular, more so with glutaraldehyde fixation, with parts of tissues consequently not responding to the lignin reaction. Post-fixation in osmium tetroxide following primary fixation in glutaraldehyde slightly improved penetration of chlorine water. However, osmium caused greater amounts of extraneous stain deposits compared with other fixative regimes. Chlorine water was confirmed as the most effective oxidising agent for reacting with groups in lignins to produce reducing residues in the Coppick and Fowler method. Sodium hypochlorite caused no reaction. Calcium hypochlorite exhibited limited oxidative capacity resulting in slight staining for lignins. The Coppick and Fowler procedure was concluded to be a suitable method for demonstrating lignins in cyto- and histochemical preparations using material fixed in either ethanol or glutaraldehyde, and with embedding in epoxy resin.     

Schistosoma mansoni: a new parenchymal cell in cercariae

A new type of cell has been identified in cercariae of Schistosoma mansoni. The perikarya (cell bodies) of these cells were located in the body (midsegment), in an area oral to the acetabulum (ventral sucker). Cytoplasmic processes extending from the perikarya ramified throughout the parenchyma of the anterior organ (oral sucker), body, and tail segments by following the path of the nerve processes from the neuropile. The perikarya of these cells had heterochromatic nuclei and a predominance of particulate material and granules (240-360 nm) in their cytoplasm. Aggregates of granules (240-360 nm) and associated vesicles (34 nm) were scattered throughout the cytoplasmic processes of the cells and formed distinct varicosed areas. These processes often connected to the tegument in the midsegment (body) of the cercariae. The granules and associated vesicles reacted (became electron dense) with fixatives reported to be detectors of biogenic amines: The glutaraldehyde/osmium tetroxide fixation procedure rendered the granules electron dense while the glutaraldehyde/chromate/osmium tetroxide fixation procedure rendered the granules and the associated vesicles electron dense. The chromate solution of the latter procedure was responsible for the electron density of the associated vesicles. The morphology of these cells (their long ramifying cytoplasmic processes) and their reaction to chromium suggests that they are probably biogenic aminergic sensory cells.     

Glutaraldehyde-fixation of yeast cells: Kinetics of a model system for enzyme cytochemistry

The kinetics of glutaraldehyde inactivation of a protoplasmic (-fructofuranosidase) and an extracytoplasmic (acid phosphatase) enzyme inSaccharomyces rouxii cells were studied at pH 5.5 and 30°C. The effects of glutaraldehyde concentration (0.5–3%), pH value, and temperature were surveyed by varying the fixation conditions. Cells from 1- to 10-day cultures retained 50–75% of their acid phosphatase activity and 15–24% of their -fructofuranosidase activity after 1-h exposures to 0.5% glutaraldehyde. The surviving -fructofuranosidase activity remained physically cryptic and was revealed only after further membrane perturbation with ethyl acetate. This crypticity barrier disappeared after overnight incubation of the treated cells at 4°C, with or without added glutaraldehyde, during which time the enzyme was resistant to further inactivation. The velocity ratio for raffinose versus sucrose, as substrate, decreased in treated cells, and changes inV _max andK _m were indicative of frank destruction of some enzyme molecules as well as modification of survivors. A comparable set of changes was also generated by treating cell-free extract with glutaraldehyde. Glutaraldehyde (0.5%) killed all yeast cells at 30°C within 5 min; at 4°C survival rates were quite high—81% after 15 min and 65% after 1 h. The bearing of these examples of enzyme inactivation, permeability barrier abolition, and structural stabilization on the general problems of yeast cytochemistry is discussed.     

Fixation, Counting, and Manipulation of Heterotrophic Nanoflagellates

Quantitative effects of several fixatives on heterotrophic nanoflagellates (HNAN) and phototrophic nanoflagellates (PNAN) were investigated by hemacytometer and epifluorescence counting techniques. Counts of Monas sp. cultures before and after fixation with unbuffered 0.3% glutaraldehyde and 5% formaldehyde showed no loss of cells during fixation, and cell concentrations remained constant for several weeks after fixation. Buffering of fixatives with borax caused severe losses, up to 100% within 2 h. Field samples from Lake Vechten showed no decline of HNAN and total nanoflagellate concentrations for at least 1 week after fixation with 5% formaldehyde and with 1% glutaraldehyde. With 1% glutaraldehyde, the chlorophyll autofluorescence of PNAN was much brighter than with 5% formaldehyde, although it was lost after a few days and thus limited the storage time of samples. However, when primulin-stained slides were prepared soon after fixation and stored at −30°C, the loss of autofluorescence was prevented and PNAN and HNAN concentrations were stable for at least 16 weeks. Effects of filtration and centrifugation on HNAN were also studied. Filtration vacuum could not exceed 3 kPa since 10 kPa already caused losses of 15 to 20%. Similar losses were caused by centrifugation, even at low speed (500 × g).     

Callose deposition at plasmodesmata

Summary The transport of ions and metabolites through plasmodesmata has been thought to be controlled at the neck region where the cytoplasmic annulus is constricted and where callose has also been localised. In order to determine the possible structural and functional effects of callose, its deposition was inhibited through incubation of the plant tissue with 2-deoxy-D-glucose (DDG) for 1 h prior to fixation in 2.5% glutaraldehyde. The inhibition of callose formation was monitored through aniline blue-induced fluorescence of callose. The neck region of the plasmodesmata fromAllium cepa L. roots treated with DDG exhibited a funnel-shaped configuration. This is in contrast to the plasmodesmata from tissue not incubated with DDG, which exhibited constricted necks similar to those previously reported. Both initial dissection and glutaraldehyde fixation induced neck constriction in plasmodesmata, however, dissection of tissue increased the frequency of constrictions. The inhibition of callose formation by chemical means showed that the neck constrictions and raised collars in this area are artefacts due to physical wounding and glutaraldehyde fixation. The external electron-dense material observed when tannic acid is included in the primary fixative appears to be unrelated to the deposition of callose at the neck region.Abbreviations DDG 2-deoxy-D-glucose     

A NEW METHOD FOR FIXATION OF UNMINERALIZED HAPTOPHYTES FOR TEM (WHOLE MOUNT) INVESTIGATIONS

A new fixation method for unmineralized haptophytes is presented. Water samples are fixed with a mixture of Lugol's solution and glutaraldehyde (final concentration 1% Lugol and 0.25% glutaraldehyde). This method preserves flagella, haptonema, and body scales very well, making light microscopical quantification at the genus level possible as well as species identification in the transmission electron microscope. The method has been tested on cultured material and natural water samples (salinities 5–34 psu).     

Techniques to preserve soluble surface components in birch pollen wall: a scanning and transmission electron microscopic study

The exine of birch pollen was examined by scanning and transmission electron microscopy in the native state and after fixation in different aqueous fixatives: glutaraldehyde + OsO4; glutaraldehyde + cetylpyridinium chloride (CPC) + OsO4; glutaraldehyde + cuprolinic blue (CB); and periodate + lysine + paraformaldehyde (PLP). The native pollen exine showed a thin (3-5-nm) border of electron-dense material lining the tectum and electron-dense material within microchannels and bacula cavities. Fixation with the addition of CPC resulted in a voluminous surface coat surrounding the pollen grain, but empty microchannels and bacula cavities. After fixation with the addition of CB, there was a thin surface coat, whereas microchannels and bacula cavities were partially filled with electron-dense material. The other fixatives led to empty microchannels and bacula cavities. There was no surface coat on the pollen grain. However, after all fixation procedures, a thin electron-dense border of the tectum remained visible. Concerning the electron-dense material filling microchannels and bacula cavities in the native pollen grain, the results obtained in the present study suggest that it is either completely lost (after conventional and PLP fixation) or, after fixation with a precipitating additive, partially (CB) or completely (CPC) solubilized and precipitated on the surface of the pollen grain as a surface coat.     

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)     

The wall associated lipoteichoic acid ofStreptococcus sanguis

A competitive ELISA is described for the measurement of lipoteichoic acid. The assay was used to determine the wall associated lipoteichoic acid ofStreptococcus sanguis which was found to represent only 2–4% of the phenol extractable content. Extracellular lipoteichoic acid was detected even after exhaustive cell washing. This material was not the result ofde novo synthesis because membrane de-polarization had no effect on the amount detected. Since extracellular lipoteichoic acid interfered with the measurement of cell surface antigen, cells were fixed with glutaraldehyde prior to assay. Lipoteichoic acid was demonstrated on the surface of fixed cells which did not leak antigen. The relevance of fixation used in antigen location studies by electron microscopy of immune-labelled cells is discussed.     

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.