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.     

Lanthanum as an intracellular stain for electron microscopy

Summary Results obtained after the normal aldehyde fixation of duodenal enterocytes for electron microscopy have been compared with results obtained when 0.1% Malachite Green or 10mm lanthanum chloride had been added during aldehyde fixation. Sections were examined without further staining, and after counterstaining with lead citrate and uranyl acetate. In unstained sections, lanthanum-treated material showed improved contrast when compared to results from the other two methods. Also, after counterstaining, areas showing excellent contrast were much more frequent and more readily detected in the lanthanum-treated material. In the microvilli of enterocytes fixed in the presence of lanthanum, the plasmalemma-glycocalyx was defined more clearly and the results were more pleasing subjectively. When Malachite Green was present in the fixative, good contrast was observed more frequently than in routinely fixed tissues, but less often than in those treated with lanthanum. It is suggested that the addition of lanthanum chloride or Malachite Green to the fixative may prove useful in many ultrastructural studies.     

Dipotassium tetramethyl osmate: a stain for electron microscopy.

Methanol solutions of dipotassium tetramethyl osmate (DTMO) have been found to be useful as general stains in electron microscopic studies of plant and fungal ultrastructure. The stain solutions are easy to prepare, stable when anhydrous and convenient to use. Although generally similar in staining to lead citrate stains, some elements of cell ultrastructure appear different with dipotassium tetramethyl osmate staining, particularly the outer cell walls of fungi. Indications of specific precipitate-producing reactions in cell storage areas are observed.     

1,3,7-Trimethylxanthine,an allelochemical from seeds ofCoffea arabica: Some aspects of its mode of action as a natural herbicide

Summary Certain aspects of selective toxicity of 1,3,7-trimethylxanthine (1,3,7-T), a natural herbicide, were studied inPhaseolus mungo andAmaranthus spinosus. Treatment of seeds ofA. spinosus with 1,3,7-T (sub-lethal concentration) caused a marked decrease (29.5%) in amylase activity. Kinetic studies with respect to substrate saturation and Km values as well asin vitro treatments of the cell-free enzyme indicated no effect on its catalytic property. The inhibitory effect of 1,3,7-T on amylase activity could not be counteracted with treatments of GA₃. A general biochemical analysis revealed that the starch/sugar ratio increased in the seedlings grown from treated seeds. Analysis also showed a higher ratio for insoluble/soluble nitrogen. It is suggested that inhibition of germination is caused by reduced synthesis of amylase which is not mediated through gibberelic acid. Treatment of seeds ofPhaseolus mungo with, 1,3,7-T. caused no change in amylase activity,in vivo nitrate reductase activity, ethanol soluble and insoluble nitrogen and protein content.     

Ring formation of perfringolysin o as revealed by negative stain electron microscopy

Perfringolysin O revealed ring- and arc-shaped structures in the absence of cholesterol by negative staining electron microscopy, while before activation with cysteine it showed indistinct arcs and irregularly curved sticks but no rings. These structures were observed only at high concentrations (more than 17 000 hemolytic units per ml) and seemed to be particle associates with 20–28 particles (about 4 nm per particle) linked in a circle. The toxin produced an inactive and high molecular weight complex in the presence of phosphotungstic acid, which was isolated by Sephadex gel filtration. These findings suggest that the rings are the toxin-phosphotungstic acid complexes produced during specimen preparation on a grid in vacuo. The toxin lost the properties necessary for ring formation though moderate modification with glutaraldehyde, showing spindle- and egg-shaped particles of about 4 nm in minor and 5 nm in major axis by negative staining. These facts suggest that the aldehyde modifies the binding sites for phosphotungstic acid, which probably are the basic groups of the toxin molecules. In the presence of cholesterol, even at a low concentration, the toxin revealed rings and arcs by negative staining and also by carbon shadowing electron microscopy, although the toxin itself did not show any characteristic structure without phosphotungstic acid. These observations suggest that the rings are the toxin-cholesterol complexes themselves. The toxin-phosphotungstic acid complexes seemed to have a structure of a single layer of particle associates, while that of the toxin-cholesterol complexes may consist of double or triple layers of the associates because its border was thicker and more distinct.     

Practical aspects of diagnostic electron microscopy.

The electron microscope technique of negative staining was first used to obtain fundamental information about virus morphology, but in recent years it has developed into a practical method for virus diagnosis. The methods employed are both simple and rapid. The following review discusses in detail the steps that must be taken to obtain good electron microscope preparations and illustrates some of the results that can be obtained.     

Chemical aspects of santalin as a histological stain

Recent research on the chemical nature of the red dyes isolated from Pterocarpus santalinus and certain West African plants, viz., Baphia nitida, Pterocarpus osun and Pterocarpus soyauxii, have been reviewed. P. santalinus contains santalins A, B and C, but no santarubin. Santalins and santarubins have been found in P. osun, P. soyauxii and B. nitida. The structural formulae of the santalins are presented and their differences from santarubins indicated. Santalins A and B have some similarities in structure with hematein. This is probably responsible for their staining properties; the possible mechanism of staining is discussed.     

The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy

Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving > 3.5 Å resolution detail in membrane proteins of modest (~ 300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function.     

C. I. acid red 52: a new stain for cells of granulocytic origin

Using the xanthene dye C.I. acid red 52 (C.I. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52. In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

Polystyrene embedding: a new method for light and electron microscopy.

Polystyrene embedments of histological specimens can be obtained with a solution of 1:14 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue, which are then glued to a Plexiglas support. Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy sections on a slide heated on a hot plate to 80 C; these can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and given acceptable results.     

New aspects of bacterial ultrastructure as revealed by modern acrylics for electron microscopy

Modern acrylics can be used over a wide temperature range (+60 degrees C to -80 degrees C) for infiltration, embedding, and polymerization. They can be used in procedures involving chemical fixation or rapid freezing. This flexibility allows for studies to be carried out upon the effects that different parameters involved in preparing biological tissue for microscopy have upon structure and retention of immunoreactivity. With most preparative methods contributions have been made to our knowledge on bacterial structure in gram-negative and gram-positive cells. The future should lie in integrating the advantages of the various methods for the purpose of advancing our understanding of bacterial structure/function.     

Alkaline lead citrate: a selective stain for glutaraldehyde precipitates of indolamines and primary catecholamines in electron microscopy.

Test-tube experiments proved that alkaline lead citrate (Reynolds, 1963), which is generally used as an electron-opaque stain, specifically reacts with precipitates formed by glutaraldehyde and biogenic amines (indolamines, primary catecholamines). Glutaraldehyde/osmium tetroxide fixation and staining of thin sections with alkaline lead citrate is recommended as an optimal preparation method of studying the above-mentioned amines at a fine structural level.