Herpetomonas samuelpessoai: electron microscopy and cytochemistry of electron-dense granules.

Herpetomonas samuelpessoai has membrane-bound electrondense granules in its cytoplasm. The electron density independs on postfixation with osmium tetroxide and is enhanced by uranyl acetate staining. The granules contain iron, have basic proteins cytochemically detected by the silver ammoniacal method, and have a peroxidase activity as detected by the diaminobenzidine method. Some of the granules also have acid phosphatase activity. It is suggested that the granules may represent either lysosomes or a storage form of tetrapyrrole derivatives which are essential for the growth and metabolism of most Trypanosomatidae.     

Osmium Tetroxide-Potassium Ferrocyanide Intensification of a Diaminobenzidine Product Obtained by Photoconversion of a Fluorescent Label: a Study of Human Neutrophil Granules

A diaminobenzidine posttreatment employing osmium tetroxide and potassium fer-rocyanide was successfully used to intensify the diaminobenzidine stain formed by photoconversion of immunofluorescent labelling. Lactoferrin labelled granules became visible following the photoconversion process. Adequate diaminobenzidine staining was obtained after irradiating the cytospin preparations with ultraviolet light for 30-40 min. The diaminobenzidine stain had advantages over the fluorescent stain owing to its greater stability, greater density, and ability to be intensified. The enhancement procedure intensified both the color and density of the diaminobenzidine product. Consequently, shorter irradiation times could be used. Osmium tetrozide solutions of 3-4% increased the intensity with minimal background staining. Ultrastructural immunogold cytochemistry on ultrathin sections confirmed the existence of the lactoferrin labelled structures observed by light microscopy of cytospin preparations indicating that these were secondary grannies. The photomicroscopy procedures used in this study were simple to perform and could be applied to studies of other cellular antigens prior to using Immnnoultramicroscopy.     

The specific cytochemical demonstration in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups

Summary A technique is described for the localization in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups. In this technique the sugar residues are oxidized by periodic acid and the resulting aldehydes condensed with pentafluorophenylhydrazine under specified conditions. Further increase in specific electron density is achieved by treating the hydrazone end-product with ammonium sulphide followed by osmium tetroxide to form an osmium black.The technique has been applied to liver and small intestine cells in which glycogen, sialomucins and sulphated mucosubstances reacted especially strongly. A marked positive reaction has also been obtained from the interstitial cell matrix and from the Golgi apparatus and multivesicular bodies of the intestinal epithelial cells.The reaction can be prevented by the omission of the periodate oxidation and, if due to glycogen, by prior treatment with diastase.     

Ruthenium red as a stain for electron microscopy. Some new aspects of its application and mode of action.

Commercial ruthenium red has been tested for its purity by spectrophotometry. Impurities detected by this method could be abolished by nitric acid-precipitation of ruthenium brown. This substance has no effect on cell surface staining and converts almost completely to ruthenium red under the conditions used in electron microscopy. It was found, by photometric analysis, that in the ruthenium red-osmium tetroxide-cacodylate combination, generally used for cell surface staining, chemical reactions between ruthenium red and osmium tetroxide occur. As aerial oxidation of hexammineruthenium2+ leads to a product with some surface staining capability, it is suggested that an oxidized product of ruthenium red is responsible for binding to cellular components, and that a reduced product of osmium tetroxide gives an additional contrast enhancement. In ruthenium red-osmium dioxide combinations ruthenium red seems to bind to cell surfaces without any molecular alteration, and contrast is gained by the model proposed by Blanquet (1976b). The latter method could open a way for investigating the binding of ruthenium red to certain natural compounds involved in calcium transport, as postulated by a number of authors. Both ruthenium-osmium combinations differ in their cell surface staining ability. The ruthenium red-osmium dioxide combination tends to form distinct subunits, whereas the osmium tetroxide variety stains homogeneously. In combination with osmium dioxide, the surface staining is affected by EDTA, and, in contrast to osmium tetroxide, a successive application of ruthenium red and osmium dioxide as possible.     

Electron microscopical demonstration of sulphated mucopolysaccharides in mouse tracheal cartilage with a diaminobenzidine-osmium tetroxide technique

Synopsis A diaminobenzidine-osmium tetroxide method for the demonstration of sulphated mucopolysaccharides has been tested at the electron microscopical level. The reaction (which involves treatment with diaminobenzidine in an acidic solution followed by oxidation with osmium tetroxide) has been carried out directly on ultra-thin sections of mouse tracheal cartilage embedded in water-soluble glycolmethacrylate. Sulphated mucopolysaccharides in the cartilage matrix were localized as a heavy, electron-dense precipitate. The method can be applied directly to sections, overcoming in this way the difficulties of penetration, and it seems to possess a higher specificity for sulphated mucopolysaccharides than that displayed by other techniques proposed previously for the ultrastructural demonstration of acid mucopolysaccharides.     

Diaminobenzidine as a Myelin Stain in Semithin Plastic Sections

A diaminobenzidine (DAB) stain for myelin in glutaraldehyde fixed, osmicated, semithin epoxy sections is described. One or 1.5 μm sections, dried onto slides, are first etched with a 1:2 dilution of saturated sodium ethox-ide:absolute ethanol, then incubated in 0.05% aqueous DAB with 0.01% hydrogen peroxide. DAB specifically stains osmium fixed myelinated nerve fibers. This permits high resolution light microscopic study of myelinated nerve fibers in semithin sections of tissues that also can be studied by electron microscopy.     

A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry.

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.     

The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells

The preservation and contrast of membranous structures in cultured cells using various postfixation procedures prior to embedding have been investigated. These include routine OsO4, ferrocyanide-reduced OsO4, osmium-thiocarbohydrazide-osmium (OTO), and ferrocyanide-reduced osmium-thiocarbohydrazide-ferrocyanide-reduced osmium (R-OTO). With standard ethanol-Epon dehydration/embedding techniques, a dramatic improvement in both membrane contrast and preservation of bilayer membrane structure was achieved using preembedding OTO in cultured cells. R-OTO yielded similar enhanced preservation and contrast of membranes. Both of these methods also resulted in an increase in the contrast of diaminobenzidine reaction product from horseradish peroxidase activity, and of lipid droplets and lipoprotein particles. However, R-OTO did not cause the same increase in the density of proteinaceous elements as was seen with the OTO method. Ferrocyanide-reduced osmium alone showed significant advantages for quantitation of immunocytochemistry using ferritin labels with bismuth subnitrate counterstain. These methods should have general usefulness for the preservation of lipid-containing structures in cultured cells.     

Immunohistochemical localization of cell surface receptors using a novel method permitting simple, rapid and reliable LM/EM correlation

Summary A method is presented which allows correlative serial section analysis by light and electron microscopy of cell surface antigens in monolayer cultures. Sites of antigenicity are shown by deposition of diaminobenzidine after pre-embedding, immunoperoxidase immunocytochemistry. Osmication is replaced by the use of gold chloride which specifically enhances the electron density of diaminobenzidine. In addition gold chloride bound to diaminobenzidine survives embedding and provides the basis for a post-embedding photochemical amplification method. Immunostained cells are embedded in LR White by a rapid technique which preserves their structure and leaves them available for subsequent post-embedding immunocytochemistry. The method is illustrated by the demonstration of epidermal growth factor (EGF) receptors on the EGF receptor-rich human carcinoma cell line A431 using a well characterized monoclonal antibody raised against EGF receptor.     

Diaminobenzidine an electron donor to photosystem 1 and to photosystem 2 in chloroplasts.

1. 3,3'-Diaminobenzidine was shown to serve as an electron donor to photosystem 1 in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In Tris-treated chloroplasts diaminobenzidine serves as an electron donor to photosystem 1 and to photosystem 2; the latter is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. 2. Addition of diaminobenzidine to Tris-treated chloroplasts causes an increase in fluorescence yield. 3. Diaminobenzidine-dependent electron transport mediated by photosystem 2 is coupled to synthesis of ATP even in the absence of an electron acceptor. This phosphorylation which is presumably supported by cyclic electron flow, is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. 4. Diaminobenzidine-dependent ATP formation, in Tris-treated chloroplasts exhibits the red-drop phenomenon. 5. The diaminobenzidine-induced cyclic photophosphorylation (mediated by photosystem 2) is resistant to a large extent to KCN-treatment which is known to inhibit reactions catalyzed by photosystem 1. On the other hand ATP formation supported by electron transport from diaminobenzidine to methyl viologen [in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea] is largely inhibited by KCN-treatment. This observation suggests that there are two coupling sites of ATP formation, one catalyzed by diaminobenzidine as a donor to photosystem 1 (in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea), and the other supported by diaminobenzidine which acts both as a donor to photosystem 2 (in Tris-treated chloroplasts) and as an acceptor (in its oxidized form) from a carrier located between the two photosystems.     

Localization of oxidized 3,3′-diaminobenzidine deposits in chloroplasts

Summary Experiments were carried out in order to localize products of diaminobenzidine in illuminated and non-illuminated isolated pea chloroplasts. The number of particles exposed by deepetching on the endoplasmic surface (ES) of the thylakoid membrane in illuminated chloroplasts was found to be higher than that of non-illuminated ones. A similar result was obtained for protrusions counted on the fractured faces of the corresponding half of the thylakoid membrane (EF). It is suggested that both the particles and the corresponding protrusions are deposits of oxidized diaminobenzidine formed in the light. These results are in agreement with biochemical experiments, indicating that diaminobenzidine donates electrons on the endoplasmic surface (ES) of the thylakoid.     

Sulphur oxidation by soil fungi including some species of mycorrhizae and wood-rotting basidiomycetes

Abstract The mycorrhizal fungi Amanita muscaria, Paxillus involutus, Hymenoscyphus ericae, Pisolithus tinctorius, Rhizopogon roseolus , and Suillus bovinus oxidized elemental sulphur to thiosulphate and sulphate in vitro. In some, but not all cases, tetrathionate was also formed. Limited oxidation of elemental sulphur by R. roseolus also occurred when growing in association with Pinus contorta in unsterilized peat. Although yeasts capable of oxidizing sulphur could not be isolated from a wide range of soils, a yeast-like fungus ( Monilia sp.) isolated from deciduous woodland soil oxidized elemental sulphur to sulphate, forming thiosulphate, but not tetrathionate. This fungus also oxidized tetrathionate to sulphate but showed only limited ability to oxidize thiosulphate to tetrathionate. Both Aspergillus niger and Trichoderma harzianum oxidized elemental sulphur in mixed culture with Mucor flavus . Larger amounts of sulphate were initially formed in mixed, compared to single culture; but by week 5 of the incubation period sulphate formation was greatest in single culture. The wood-rotting fungi, Hypholoma fasciculare and Phanerochaete velutina showed a limited ability to oxidize elemental sulphur in vitro but were incapable of oxidizing the element when growing as mycelial cords in non-sterilized soils. The relevance of these results to the possibility that fungi play a role in sulphur oxidation in soils is commented upon.     

Electrochemical DNA sensing using osmium complexes as hybridization indicators

A surface-based method for the study of the interactions of DNA with redox-active osmium complexes is described. The study was carried out using gold electrodes modified with DNA by adsorption and [Os(bpy)3]3+/2+ (bpy=2,2'-bipyridyl) or [Os(phen)3]3+/2+ (phen=1,10-phenantroline) as electrochemical indicators. The method, which is simple and reagent saving, allows the accumulation of osmium complexes on the DNA layer. The amount of osmium complex bound by the layer of double-stranded (dsDNA) or single-stranded DNA (ssDNA) adsorbed at gold electrodes was estimated from the cyclic voltammetric (CV) peak charge of osmium complex reduction. The dissociation constants (K) for the oxidized and reduced forms of a bound species are also estimated. [Os(phen)3]3+/2+ was applied to a probe for electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 70 pmol/cm2 on a gold electrode. Following hybridization with the complementary DNA, the osmium complex was electrochemically accumulated on the double-stranded DNA layer and the differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of [Os(phen)3]3+/2+ that was bound to the double-stranded DNA on the electrode.     

Electron-dense material in the cell sall/plasma membrane interface of specialized cortical cells of peanut nodules.

The nitrogen-fixing peanut root nodules have 2-3 layers of specialized cortical cells surrounding the infected zone. These cells are morphologically distinct from the other cortical cells due to the presence of prominent amyloplasts, lipid bodies (oleosomes), large microbodies and localized pockets of electron-dense material at the plasma membrane-cell wall interface. The material is resistant to solubilization by hexane and chloroform. Electron density is enhanced by p-phenylenediamine staining and heavy metal ions such as iron, uranium and osmium. The silver proteinate staining method for suberin has shown positive results. Peroxidative activity can be demonstrated by diaminobenzidine reaction. The material appears to be proteinaceous, but could be complexed with suberin or phenolics. Although the exact nature of this material remains to be clarified, its unique position in the cells of specialized tissue of the cortex relates it to the regulation of bidirectional transport of gases and solutes during symbiosis in the root nodules.     

The use of chromic potassium sulphate in bone electron microscopy

The ultrastructure of endochondral bone was studied using an aqueous solution of chromic potassium sulphate as the decalcifying agent. 0.5 mm thick sections of rat tibiae were fixed in buffered glutaraldehyde, immersed in an aqueous solution of 1% chromic potassium sulphate pH 3.4, dehydrated and embedded in Poly Bed 812 without exposure to osmium tetroxide. In unstained sections we observed clusters of crystal like structures throughout the osteoid and calcifying cartilage matrix as well as solitary needle shaped structures in association with collagen fibrils. Stained sections revealed nuclei, endoplasmic reticulum, membrane limited dense granules, mitochondrial particles and other cell components typical of bone cells. It appeared that the chromic potassium sulphate method preserves the relationship between hard and soft tissues well, gives fine cytological detail and produces images of intracellular and extracellular deposits identical to untreated crystallites. It is concluded that the chromic potassium sulphate method is indicated for ultrastructural studies of bone.     

Osmium-labeled polynucleotides. Incorporation of additional heavy atoms (mercury) via ligand substitution reactions

The reaction of osmium tetroxide with biological materials, particularly nucleic acids, has considerable utility in electron microscopy and X-ray crystallography. This heavymetal label introduces an electrophilic center which may serve as a means of attachment of nucleophiles. Nucleophilic ligand substitution reactions have been exploited as a means of adding more heavy metals (mercury) onto the osmium label. Furthermore, the technique is quite general and could be used to modify the osmium label with, for example, fluorescent groups. The nucleophilic exchange reactions have also been studied using ¹H nmr spectroscopy with a representative heterocyclic osmate ester derivative, the bis(pyridine) osmate ester derivative of TMP. These studies have defined the nature of the ligands which lead to stable osmium labels.     

Sensitive detection of hybridocytochemical results by means of reflection-contrast microscopy

A new sensitive method for visualization of nonautoradiographic hybridization results in microscopic preparations is described. The method is based on the reflection of the incident light by diaminobenzidine precipitates deposited at the site of hybridization during an indirect hybridocytochemical procedure. The reflected light is detected by means of reflection-contrast microscopy. The applicability of the procedure is demonstrated with nucleic acid probes modified with 2-acetylaminofluorene groups. These in turn are localized in situ by an indirect immunoperoxidase reaction. Besides its sensitivity, this simple visualization technique possesses the additional advantages, over absorption and fluorescence microscopy, that it provides a total DNA counterstain and a chromosomal banding pattern.     

Oxidation of pyrimidine nucleosides and nucleotides by osmium tetroxide

1. Pyrimidine nucleosides such as thymidine, uridine or cytidine are oxidized readily at 0° by osmium tetroxide in ammonium chloride buffer. There is virtually no oxidation in bicarbonate buffer of similar pH. Oxidation of 1-methyluracil yields 5,6-dihydro-4,5,6-trihydroxy-1-methyl-2-pyrimidone. 2. Osmium tetroxide and ammonia react reversibly in aqueous solution to form a yellow 1:1 complex, probably OsO₃NH. A second molecule of ammonia must be involved in the oxidation of UMP since the rate of this reaction is approximately proportional to the square of the concentration of unprotonated ammonia. 3. 4-Thiouridine reacts with osmium tetroxide much more rapidly than does uridine. The changes of absorption spectra are different in sodium bicarbonate buffer and in ammonium chloride buffer. They occur faster in the latter buffer and, under suitable conditions, cytidine is a major product. 4. Polyuridylic acid is oxidized readily by ammoniacal osmium tetroxide, but its oxidation is inhibited by polyadenylic acid. Pyrimidines of yeast amino acid-transfer RNA are oxidized more slowly than the corresponding mononucleosides, especially the thymine residues. Appreciable oxidation can occur without change of sedimentation coefficient.     

Cytochemical localization of catalase in leaf microbodies (peroxisomes)

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3''-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.     

Post-embedding localization of glycoconjugates by means of lectins on thin sections of tissues embedded in LR white

Summary A simple post-embedding technique for the electron microscopical detection of lectin-binding sites using thin sections of tissues embedded in the resin LR White is described. With this technique, no prior etching of the sections is necessary. The cellular fine structure is well preserved and permits close correlation of the labelling to distinct cellular compartments. After mild aldehyde fixation (4% formaldehyde and 0.5% glutaraldehyde for 30 min), enterocyte brush border, vesicles and lysosomes as well as goblet cell Golgi apparatus and mucin are intensely stained after 30–60 min.The hydrophilia and penetrability of LR White is shown by the formation of oxidized diaminobenzidine reaction product arising from horseradish peroxidase-conjugated lectins. The precipitate not only covers the surface of the sections but is also formed within the resin, as is revealed on cross-sections through thin and semithin sections. The addition of 0.2m solutions of the appropriate inhibitory sugars prevented staining, which indicates a specific binding. Examples are given of the binding of gold-, ferritin- and peroxidase-conjugated lectins for the purpose of detecting glycoconjugates in various intracellular compartments.