Transient holes in the erythrocyte membrane during hypotonic hemolysis and stable holes in the membrane after lysis by saponin and lysolecithin

Ferritin and colloidal gold were found to permeate human erythrocytes during rapid or gradual hypotonic hemolysis. Only hemolysed cells contained these particles; adjacent intact cells did not contain the tracers. Ferritin or gold added 3 min after the onset of hypotonic hemolysis did not permeate the ghost cells which had, therefore, become transiently permeable. By adding ferritin at various times after the onset of hemolysis, it was determined that for the majority of the cells the permeable state (or interval between the time of development and closure of membrane holes) existed only from about 15 to 25 sec after the onset of hemolysis. It was possible to fix the transient "holes" in the open position by adding glutaraldehyde only between 10 and 20 sec after the onset of hemolysis. The existence of such fixed holes was shown by the cell entry of ferritin and gold which were added to these prefixed cells. Membrane defects or discontinuities (of the order of 200–500 A wide) were observed only in prefixed cells which were permeated by ferritin subsequently added. Adjacent prefixed cells which did not become permeated by added ferritin did not reveal any membrane discontinuities. Glutaraldehyde does not per se induce or create such membrane defects since cells which had been fixed by glutaraldehyde before the 10-sec time point or after the 180-sec time point were never permeable to added ferritin, and the cell membranes never contained any defects. It was also observed that early in hemolysis (7–12 sec) a small bulge in one zone of the membrane often occurred. Ghost cells produced by holothurin A (a saponin) and fixed by glutaraldehyde became permeated by ferritin subsequently added, but no membrane discontinuities were seen. Ghosts produced by lysolecithin and fixed by glutaraldehyde also became permeated by subsequently added ferritin, and many membrane defects were seen here (about 300 A wide).     

Staining of xylem parenchyma mitochondria with photo-oxidized 3,3'-diaminobenzidine

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.     

Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO4) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with glutaraldehyde only, but seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO4 increases UAc uptake by a factor of about 3. The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO4; similar levels for final PTA uptake are also observed. An increase in the resin content of embedded chromatin postfixed with OsO4 is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

The effect of chemical fixatives on cell walls of Bacillus subtilis.

Cell walls of Bacillus subtilis were treated with several chemical fixatives which are commonly used preparatory to electron microscopy; i.e., osmium tetroxide, formaldehyde, acrolein, crotonaldehyde, and glutaraldehyde. Dimensional analysis was performed on thin sections of fixed walls from plastic embeddings and, by means of the statistical technique of multiple comparisons, significant differences were found between wall thicknesses from the various fixations. These differences varied with the fixation time and the type of fixative used in the reaction. When compared to embedded walls which had been stained before fixation, the overall effect was a reduction in wall thickness which was attributed to fixative action and not to the embedding or staining processes. The reduction of wall thickness was even more apparent when dimensions of fixed walls were compared to published dimensions of both frozen sections and freeze-etch profiles. Since these fixatives bind to reactive sites within the wall fabric, a change in electrochemical charge density is effected which can be monitored in terms of heavy-metal-binding capacity. Most monoaldehyde fixatives and osmium tetroxide render the wall as reactive, or less reactive, to uranyl acetate as unfixed walls, whereas glutaraldehyde can significantly increase the binding capacity.     

Use of confocal microscopy in examining fungi and bacteria in wood

When used in conjunction with digital image processing techniques, confocal laser scanning microscopy (CLSM) enables non-invasive optical sectioning, allowing micromorphologies of wood decay to be examined at any depth within a relatively thick (0.05-0.1 mm) wood specimen without incision. In this study, the use of specially tailored multi-fluorescent staining techniques with CLSM produced new information concerning spatial relationships between fungi and bacteria and the wood substrate, particularly in regard to their 3D characteristics. Glutaraldehyde fixation and a chitin fluorescent probe were used to locate fungal hyphae in wood. Bacteria colonising wood were examined using a fluorescent phospholipid probe. By counterstaining wood with this probe and a fluorescent dye specific for Gram-positive bacteria, it was possible to clearly distinguish Gram types through simultaneous, multichannel fluorescent CLSM imaging. The combination of glutaraldehyde fixation and phospholipid probing proved to be reliable for detecting wood-degrading bacteria in wood cell walls.     

Observations on the Kinetics of Uranyl Acetate and Phosphotungstic Acid Staining of Chromatin in thin Sections for Electron Microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO₄) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with gutaraldehyde only, but stain uptake is reduced following a long wash with distilled water to a level similar to that seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO₄ increases UAc uptake by a factor of about 3.

The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO₄; similar levels for final PTA uptake are also observed.

An increase in the resin content of embedded chromatin postfixed with OsO₄ is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

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.     

Electron microscopic study of the role of lipid micelles in intestinal fat absorption

In vitro micellar solutions of oleic acid, monoolein, and sodium taurocholate were studied electron microscopically. They contained osmiophilic particles 30-200A in diameter. Osmium staining alone was sufficient to demonstrate the particles; lead staining had little effect on their appearance. The intestinal intraluminal contents from rats during the absorption of unsaturated fat also contained osmiophilic particles, 40-200A, and numerous similar particles were found between the microvilli and engaged in the fine filamentous coating of microvilli. In the lumen only, larger emulsion-type droplets were also seen. The small particles were demonstrable in osmium-fixed material, with or without lead stains, and staining with lead only increased contrast of the particles. Spherules measuring about 1000A in diameter with walls about 100A thick were observed in the terminal web during fat absorption, at which time they were slightly larger and more numerous than in fasting rats. It is proposed that during fat absorption micellar particles are engaged in the filamentous material covering the microvilli and then enter the absorptive cell either by molecular diffusion across the plasma membrane or by being incorporated into the walls of thick-coated spherules which then pass into the subapical cytoplasm.     

Loss of antibody binding to prefixed cells: Fixation parameters for immunocytochemistry

Summary The denaturing effects of various types of fixative solutions on 5 cell surface antigens on mouse T-lymphocytes (Thy-1, T-200, Lyt-1, Lyt-2, and Th-B) were studied. For this purpose, cells were fixed with paraformaldehyde, glutaraldehyde, acrolein and osmium tetroxide at various concentrations. Fixed cells were then incubated with monoclonal antibodies and appropriate second stage antibodies or conjugates. The degree of antibody binding to these cells was determined quantitatively using flow-cytometry with a fluorescence-activated cell sorter or with a semi-automatic micro-ELISA system. The data obtained indicate that paraformaldehyde and glutaraldehyde preserve all five tested antigen molecules, whereas antibody binding to cells fixed in acrolein and osmium tetroxide is rapidly reduced at increasing concentrations of the fixative. The optimal concentration of paraformaldehyde is in the range 0.5–1%, whereas glutaraldehyde should be used at concentrations between. 0.05 and 0.1%. Cells fixed with 0.5% paraformaldehyde or with 0.05% glutaraldehyde are stable and can be stored for at least one week prior to incubation with antibodies.     

Osmium dependent argentaffin staining of lysosomes

Summary Lysosomes stain with the argentaffin reaction after fixation with glutaraldehyde followed by osmium tetroxide. The reaction works well both at the level of the light and electron microscope. Control experiments show that this argentaffinity is caused by reduced osmium tetroxide. No staining could be observed in freeze-dried material, in tissues fixed only with glutaraldehyde, or after bleaching of the sections with hydrogen peroxide solutions. In the electron microscope, the population of lysosomes appears heterogeneous as related to the density of silver deposits over the organelles. No correlation is found between size and argentaffinity of lysosomes. X-ray microanalysis of sections from glutaraldehyde/osmium tetroxide fixed material reveals significantly higher amounts of osmium in lysosomes, as compared to other cell organelles (e.g. peroxisomes or mitochondria). A significant peak for silver is observed in lysosomes after treatment of the sections with ammoniacal silver solution, whereas the signal for osmium is reduced. Amounts of sulphur are too low to be detected in lysosomes. It is concluded that argentaffin staining of lysosomes is an osmium dependent reaction.Parts of these results have been presented as a poster during the 20th Congress of Electron Microscopy, joint session of the Austrian Society of Electron Microscopy and the German Society of Electron Microscopy, August 23–28, 1981, Innsbruck, Austria     

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about one-third those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried material fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

Summary A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about onethird those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried metarial fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Presence and cellular distribution of soybean agglutinin-binding epitopes in two strains of<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The cellular location ofN-acetylgalactosamine inBacillus subtilis strains 168 and 170 was examined by electron microscopy using gold-conjugated soybean agglutinin (SBA) as a marker. Post-embedding labeling of sectioned material showed SBA-reactive, galactosamine-containing polymers associated with the cell membrane or the cytoplasm of the two strains. This intracellular location was distinct from the concanavalin A-binding epitopes that were located over the cell wall. Labeling of whole cells (native, fixed in glutaraldehyde, or treated with proteinase K, or Tween 20) before negative staining revealed no galactosamine exposed on the surface of strain 168. On the surface of strain 168 some exposed galactosamine terminal residues were detected; their accessibility to SBA increased when Tween 20 or proteinase K was applied.     

Immunocytochemical localization of insulin-like immunoreactivity in mouse seminal vesicle

Insulin-like immunoreactivity was localized in tissue sections and cell cultures of mouse seminal vesicle using the indirect technique of immunocytochemistry. Seminal vesicles were cut into fragments, fixed in 2.5% glutaraldehyde, embedded in epoxy resin, sectioned at 1 micron, and transferred to glass slides. Epithelial cell cultures of seminal vesicle were grown on coverslips in Dulbecco's Minimal Essential Medium for 4-6 days and fixed in 2.5% glutaraldehyde. Sections (etched with sodium ethanolate) or coverslips were incubated in guinea pig antiporcine insulin antiserum, in antiserum immunoabsorbed with porcine insulin, or in normal guinea pig serum. For indirect immunocytochemistry, incubation with primary antiserum was followed by treatment with rabbit anti-guinea pig immunoglobulin (Ig) G conjugated to peroxidase, or with protein A and then rabbit peroxidase anti-peroxidase (PAP). Finally, treated samples were incubated in phenylenediamine-pyrocatechol-H2O2 substrate mixture for 6-8 min at room temperature. Specific immunoreactivity to insulin antisera was confined to the epithelium of the seminal vesicle in tissue sections. No staining occurred in subepithelial connective tissue. Specific immunoreactivity was also observed in the cytoplasm of cultured seminal vesicle epithelial cells.     

A Comparison of Substrates for Human Umbilical Vein Endothelial Cell Culture

We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time.

Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.     

An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml(-1)) followed by achromopeptidase (60 U ml(-1)) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.     

Cytochemical and X-ray microanalytical studies of calcium in the extending zone, particularly in the rhizodermis of Zea mays roots

Calcium was detected in CaCl (10 m M )-pretreated roots of Zea mays L. (cv. LG 11) by means of electron probe X-ray microanalysis with energy-dispersive spectrometry (EDS) using embedded samples, fixed by the antimonate staining procedure. A high level of calcium was found where large amounts of antimonate precipitates were observed by light or transmission electron microscopy. In the elongation zone, after 20 h in humid air following a 2 h CaCl₂ pretreatment, the level of calcium was higher in trichoblasts than in atrichoblasts. In these cells it was detected mainly in the walls and nucleus, and antimonate staining was observed in the walls. Abundant precipitates containing calcium were associated with the nucleus, vacuoles, mitochondria and endoplasmic reticulum of non-differentiated cells, whereas they were confined to the walls of these cells just after the CaCl₂ pretreatment. The involvement of calcium in the formation of root hairs is discussed.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Some unusual staining properties of tannic acid in plants

Summary Maize root tips were fixed in glutaraldehyde fixatives containing tannic acid and then processed for electron microscopy. Under these conditions, tannic acid selectively stained the contents of the Golgi apparatus secretory vesicles of some outer root cap cells, the cell walls of all cells, and substances in, and adjacent to, intercellular connections of mature primary walls and of secondary walls. Intercellular connections of the young primary walls were not stained. Plasma membranes, and substances associated with the outer leaflets of the plasma membranes, were also stained. Tannic acid-positive material was associated with the cell plate vesicles of forming walls but very little, or none, was associated with the Golgi apparatus vesicles of dividing cells.     

Ultrastructure of rumen bacterial attachment to forage cell walls.

The degradation of forage cell walls by rumen bacteria was investigated with critical-point drying/scanning electron microscopy and ruthenium red staining/transmission electron microscopy. Differences were observed in the manner of attachment of different morphological types of rumen bacteria to plant cell walls during degradation. Cocci, constituting about 22% of the attached bacteria, appeared to be attached to degraded plant walls via capsule-like substances averaging 58 nm in width (range, 21 to 84 nm). Many bacilli appeared to adhere to forage substrates without distinct capsule-like material, although unattached bacteria with capsules were observed occasionally. Certain bacili appeared to be attached to degraded tissue via small amounts of extracellular material, but others apparently had no extracellular material. Bacilli with a distinct morphology due to an irregularly folded, electron-dense outer layer or layers (about 15 nm thick) and without fibrous extracellular material consituted about 37% of the attached bacteria and were observed to adhere so closely to degraded plant walls that the bacterial shape conformed to the shape of the degraded zone. In the rumen ecosystem, bacteria appeared to adhere to plant substrates during degradation by capsule-like material and by small amounts of extracellular material, as well as by the other means not observable by electron microscopy.