A Urea Silver Nitrate Method for Nerve Fibers and Nerve Endings

A silver nitrate stain for nerve fibers and endings applicable to paraffin sections on the slide utilizes the properties of urea to accelerate the procedure and improve the specificity of the stain. After removal of the paraffin the sections are run through absolute, 95% and 80% alcohol and placed for 60-90 minutes at 50-60°C. in: 1% aqueous silver nitrate, 100 ml.; urea, 20-30 g.; 1g. mercuric cyanide and 1 g. picric acid in 100 ml. of distilled water, 1-3 drops. After the silver bath they are rinsed quickly in 2 changes of distilled water and reduced for 3-5 minutes at 25-30°C. in: water, 100 ml.; sodium sulfite, anhydrous, 10g.; hydroquinone, 1-2g.; urea, 20-30g. They are then washed thoroughly in 4-5 changes of distilled water, passed through graded alcohols into 80% alcohol and examined under the microscope. If nerve fibers are not distinct, the sections are returned to the same urea-silver-nitrate bath for 10-15 minutes, rinsed, reduced, washed and dehydrated as before. This process may be repeated until staining is adequate; then they are dehydrated, cleared, and mounted.

Nerve fibers show a color range from brown to black; nerve cells from yellow to brown; and the background, depending on the type of tissue and its fixation, from yellow to light brown.     

Kinematographische Dokumentation einer amitotischen Zellteilung

Summary Identification of argyrophilic cells, in pancreatic islets of normal rabbits, is accomplished by light and electron microscopy in osmium-fixed plastic-embedded tissues.Fixative, pretreatment and pH of silver nitrate solution were essential for the light microscopic study to reveal argyrophilic cells in osmium-fixed plastic-embedded pancreatic islet tissue. The best result was obtained with Dalton's osmium fixation and buffered silver nitrate methanamine solution at pH 9.O. The cytoplasmic granules of argyrophilic cells generally are densely packed but some of the cells show only sparse silver impregnated granules in the cytoplasm. Occasionally there are some non-argyrophilic granular cells in which, after silver impregnation, the cytoplasm appears clear. There are three kinds of cells in the pancreatic islets, i.e., argyrophilic granular cells, non-argyrophilic granular (clear) cells, and beta cells (situated centrally in the islet and stained light yellow in silver impregnated sections).The cells known as argyrophilic cells in light microscopy can be identified as alpha cells in electron micrographs by comparison of consecutive sections of the same cell.The author would like to express his appreciation to professor Roy C. Swan for his generous guidance.     

Schistosoma mansoni: localization of calcium-detecting reagents in electron-lucent areas of specific preacetabular gland granules

In an attempt to establish the exact location of calcium within the preacetabular glands of cercariae of Schistosoma mansoni, these larvae were exposed to reagents (potassium oxalate, potassium pyroantimonate, chloranilic acid, and silver nitrate) useful in the detection of calcium, and were subsequently observed with the aid of light and electron microscopes. Cercariae incubated in potassium oxalate and viewed in polarized light showed birefringence only in the preacetabular gland funduses. At the ultrastructural level, the preacetabular glands of potassium oxalate-treated cercariae had no electron-dense precipitate, but instead had translucent, irregularly shaped inclusions, similar to spaces left by volatilized calcium oxalate as described by others. Pyroantimonate treatment, on the other hand, localized the reaction in the electron-lucent areas of the light-spotted granules. The von Kossa silver nitrate procedure destroyed the secretory granules; therefore, an electron-dense precipitate was distributed throughout the gland. However, pretreatment with chloranilic acid before fixation preserved the granules, and subsequent exposure to the von Kossa silver nitrate gave a reaction identical to that obtained with the pyroantimonate alone. When viewed in polarized light, chloranilic acid-incubated cercariae showed birefringence in the fundus and duct areas.     

Histochemical Differentiation of Chloride from Other Ions Precipitated by Silver Nitrate in Freeze-Substitution Fixation

The method is based on substitution fixation at —25° C of quickly frozen tissue with a 90% alcohol solution saturated with silver nitrate. The silver salts are photochemically reduced in the histological preparations. At this low temperature very little staining of the protein structure of the tissue takes place. Silver ions adsorbed by the tissue can be removed by treatment with a sodium nitrate solution. About 2/3 of the brown material in the histological preparations of cerebral cortex was due to the chloride in the tissue, 1/6 to the phosphate, 1/10 to an unidentified (probably organic) anion, and 1/20 to bicarbonate. When the alcoholic silver nitrate solution used for the fixation is acidified, or the sections are treated with nitric acid, the colored material consists of reduced silver chloride only. A comparison of the light absorption in histological preparations of cortex treated with neutral and with acid solutions supported the conclusion that about 2/3 of the colored material in the tissue is reduced silver chloride.     

A silver-impregnation-toluidine blue technique for demonstration of embryonic skeletal structures in paraffin sections

Summary Dewaxed and hydrated sections are treated with 1% aq. silver nitrate in bright light for 60 min and then thoroughly washed with distilled water followed by a rinse in Kolthoff's buffer (pH 3.3). The sections are next treated with 0.25% toluidine blue dissolved in buffer for 3 min, rinsed in buffer, allowed to dry and then cleared and mounted. Foci of calcification appear black and cartilage-matrix lilac to deep mauve. Nuclei appear deep blue in contrast to the pale blue of the cytoplasm.     

Oxalate pretreatment and use of a physical developer render the Kossa method selective and sensitive for calcium

Summary Based on experiments on agarose gels and tissue, a procedure has been developed which greatly improves the sensitivity and the specifity of the Kossa method for demonstrating calcium in tissue. Tissue calcium is immobilized by acetonic oxalic acid, which simultaneously removes the other sorts of anions capable of precipitating silver ions (e.g. phosphate, carbonate). The resulting submicroscopic grains of calcium oxalate are converted first into silver oxalate then into metallic silver by a treatment with silver nitrate followed by an ultra-violet irradiation (Kossa reaction). These submicroscopic metallic silver grains are enlarged up to microscopic visibility by means of physical development, which makes the staining highly sensitive. Costaining of the argyrophil sites in the tissue is totally suppressed by various tricks, which render the silver staining selective for calcium.     

Oxalate pretreatment and use of a physical developer render the Kossa method selective and sensitive for calcium

Based on experiments on agarose gels and tissue, a procedure has been developed which greatly improves the sensitivity and the specifity of the Kossa method for demonstrating calcium in tissue. Tissue calcium is immobilized by acetonic oxalic acid, which simultaneously removes the other sorts of anions capable of precipitating silver ions (e.g. phosphate, carbonate). The resulting submicroscopic grains of calcium oxalate are converted first into silver oxalate then into metallic silver by a treatment with silver nitrate followed by an ultra-violet irradiation (Kossa reaction). These submicroscopic metallic silver grains are enlarged up to microscopic visibility by means of physical development, which makes the staining highly sensitive. Co-staining of the argyrophil sites in the tissue is totally suppressed by various tricks, which render the silver staining selective for calcium.     

Enzyme histochemical demonstration of alkaline phosphatase activity in plastic-embedded tissues using a Gomori-based cerium-DAB technique

We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.     

Visualization of Legionella Pneumophila in Paraffin Sections using Hexamine Silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 μm. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.     

A New Technique for Localization of Cellulase in situ Using Silver Nitrate

A new staining technique has been developed for the histochemical localization of cellulase in plant tissues by light microscopy. The products of cellulolysis are reducing sugars which can reduce the salts of heavy metals under appropriate conditions. The present technique relies on the deposition of black silver oxide due to reduction of alkaline silver nitrate to detect cellulase in tissues.     

Histochemical investigations on the nature of large blood vessel endothelial and medial argyrophilic lines and on the mechanism of silver staining

Summary A study of the mechanisms involved in silver staining of blood vessels has been performed on the rabbit and rat aorta and vena cava, both in fixed and unfixed states. Pretreatment with cationic detergents, organic solvents, and solutions containing free iodide ions inhibited the silver staining. Anionic or neutral detergents, oxidizing agents, binders of such ions as Ca⁺⁺, Mg⁺⁺ and SO ₄ ^- failed to inhibit the staining. Staining of the intercellular gaps between endothelial cells and between smooth muscle cells could also be obtained if vessels were treated with a cationic detergent and bromocresol green, or by a modified Hale's colloidal iron technique. Silver lines could be returned to dechlorinated vessels, if treated with sodium chloride before silver nitrate staining, but not vice versa; by an extended treatment with dilute silver nitrate or with gold chloride following normal silver nitrate staining; and by treatment with heparin prior to silver staining. Dark chamber experiments have demonstrated that a photographic developer can take the place of light in the silver staining procedure and that a photographic fixer has the same effect on vessel silver staining as dechlorination.The obtained results have led to the hypothesis that silver staining of vessels occurs in two stages. In the first silver ions from silver nitrate are bound by polyanions located primarily in the intercellular gaps, and then reduced. This produces a network of reduced silver grains which, however, are still too sparsely aggregated to be visualized. Chloride ions in the tissues also bind and precipitate silver ions preventing their removal in subsequent rinsing procedures. In the second stage light (or a photographic developer) reduces the silver ions in silver chloride, producing a visible accumulation of metallic silver, but only around the silver grains reduced during the first stage, analogous to the photographic process.The possible existence and function of an intercellular cement substance is discussed in light of the evidence for the presence of polyanionic groups in the intercellular gaps.     

A silver method for counterstaining plastic embedded tissue

This report presents a method which can be used for counterstaining semithin sections of plastic embedded tissue. The sections are treated with a solution of silver lactate, followed by physical development. During the silver lactate treatment, silver ions are bound by various tissue components as metallic silver or silver sulfide. During physical development catalytic reduction of silver ions to metallic silver takes place where silver has been bound in the tissue, enlarging the silver deposits to microscopically visible dimensions. The amplified silver deposits give high contrast staining in yellow, brown and black suitable for both color and monochrome photography. The localization of the silver deposits is highly specific and may reflect several independent chemical processes. Examples in several tissues are shown.     

A Silver Method for Counterstaining Plastic Embedded Tissue

This report presents a method which can be used for counterstaining semithin sections of plastic embedded tissue. The sections are treated with a solution of silver lactate, followed by physical development. During the silver lactate treatment, silver ions are bound by various tissue components as metallic silver or silver sulfide. During physical development catalytic reduction of silver ions to metallic silver takes place where silver has been bound in the tissue, enlarging the silver deposits to microscopically visible dimensions. The amplified silver deposits give high contrast staining in yellow, brown and black suitable for both color and monochrome photography. The localization of the silver deposits is highly specific and may reflect several independent chemical processes. Examples in several tissues are shown.     

Reprecipitation during the preparation of demineralized sections. II. Experimental observations.

Teeth were incompletely demineralized by immersion in unchanged 10% formic acid for 7 days. Reprecipitation deposits of secondary calcium phosphate were present in the dentin and soft tissues of the dental pulp and, if the final pH was 3 or greater, in the remnants of the periodontal ligament. The deposits in the dentin appeared to be intratubular. Deminieralized sections of teeth suspended in supersaturated solutions of brushite contained similar deposits in the soft tissues. It is suggested that reprecipitation of secondary calcium phosphates is a frequent intermediate stage during demineralization with formic acid.     

Visualization of Legionella pneumophila in paraffin sections using hexamine silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 microns. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.     

On the origin of photophosphorylation

A model based on quinol phosphates is proposed for the origin of photophosphorylation. This model is divided into three time periods. In the early period, when the primitive earth was under reducing conditions, quinol phosphates were produced through quinol radical intermediates formed by the activation of hydroquinones with ultraviolet light. Phosphorylation of a number of acceptor molecules including inorganic orthophosphate and adenosine diphosphate occurred when quinol phosphate was oxidized by Fe⁺³ or a water soluble iron-sulfur complex. After the appearance of a rudimentary ozone layer (middle period), ultraviolet light was no longer an important factor in primordial chemistry. Quinol phosphates were then produced by visible light activation of porphyrin-quinone charge transfer complexes. In the presence of light, electrons from H₂S, H₂ and several reduced organic compounds were transfered through the porphyrin to quinone yielding the quinol radical. Again, quinol phosphate was produced from breakdown of the free radical. Phosphorylation of a number of acceptor molecules was achieved when quinol phosphates were oxidized by the iron-sulfur complexes. Evolutionary pressure to increase the efficiency of these reactions resulted in the electron donor-porphyrin-quinone-iron-sulfur complex becoming more lipophilic and thus associated with the protomembrane of the evolving protocell. In the late period the protomembrane became more sophisticated and quinone was replaced as the primary electron acceptor in the photoprocess by one of the iron-sulfur complexes originally present as oxidizing agents for the quinol phosphates. Quinones eventually lost their role as phosphorylating agents and became only electron and proton shuttles in the evolving electron transport chain. The protocell evolved the ability to use water as the electron donor as the relative roles of iron and quinone in the photoprocess switched.     

Light microscopical demonstration of non-specific alkaline phosphatase activity with an incubation medium containing cerium and two calcium as the capturing agents. The cerium/calcium-hydrogen peroxide-P-phenylenediamine/pyrocatechol (Ce/Ca-H2O2-PPD/PC) double capture technique.

A new method for the light microscopical demonstration of alPase activity in cryotome sections by using simultaneously cerium and calcium as capturing agents (double capture technique) is described. This method has an increased sensitivity compared with the single cerium-based and the Gomori based-cerium (single calcium and cerium converted) with techniques described previously. Presuming that the enzymatic activity during incubation of sections in the presence of a defined capturing agent is constant, the increased sensitivity after employment of the double capture method could be attributed to a decrease of enzyme inhibition by cerium through the presence of calcium. Based on model experiments it is assumed that calcium phosphate and cerium phosphate are the primary reaction products, the former converting into cerium phosphate already during incubation. The remaining calcium phosphate is converted completely by treatment with cerium citrate solution (conversion reaction). After oxidation with H2O2 the cerium perhydroxyphosphate was visualized in a paraphenylenediamine/pyrocatechol (Hanker-Yates reagent) solution without H2O2 to give a black reaction product. This visualization procedure is superior to the DAB or DAB-Ni mode as published earlier. Some results concerning the mode of inhibition of the pseudoperoxidase activity of the hemoglobin are presented.     

The mechanism of calcium uptake by liver microsomes: effect of anions and ionophores

The mechanism of calcium uptake by liver microsomes was investigated using various anions and ionophores. Calcium uptake was shown to be specific to microsomes and unlikely to be due to contamination by plasma membranes by correlation of calcium uptake to the marker enzymes specific for these two fractions. Under the conditions employed, phosphates, sulfate, chloride, acetate, nitrate, thiocyanate, maleate, succinate and oxalate all stimulated calcium uptake by microsomes, but to different degrees. The greatest effect (4-6-fold) was observed with phosphate. On the contrary, phosphate is the only anion that stimulates the plasma membrane calcium uptake to any significant degree. Treatment of isolated microsomes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) resulted in inhibition of ATP- and anion-dependent calcium uptake. A lipid-permeable organic acid such as maleate retained its ability to promote calcium uptake in DIDS-treated microsomes. However, a lipophilic anion, such as nitrate, stimulated calcium uptake only in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). In addition, 2 microM valinomycin, when added in the absence or presence of 10 to 100 mM K+, had no stimulatory effect on calcium uptake. These results appear to be consistent with a model in which the active uptake of calcium into microsomes involves electroneutral Ca2+-nH+ exchange.     

Isolation of a starch utilizing, phosphate solubilizing fungus on buffered medium and its characterization

A phosphate solubilizing fungus, Paecilomyces marquandii AA1 was isolated from phosphate deficient soil on Pikovskaya's medium buffered with Tris-HCl pH 8. The organism was identified on the basis of morphological characterization and by sequencing of 18S rRNA gene. The organism could release phosphate from both buffered and unbuffered medium and solubilized rock phosphates from various places. The effect of concentration of ore, temperature, carbon and nitrogen sources on solubilization of rock phosphate was studied. Ammonium salts were the best nitrogen source, followed by asparagine, sodium nitrate, potassium nitrate, urea and calcium nitrate in that order.     

Opposite staining effect of two silver-staining techniques on sister chromatids

Opposite differential staining between sister chromatids was obtained by two silver-staining techniques on chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) and pretreated with Hoechst plus black light. Both silver-nitrate and silver-carbonate staining were affected by chemical extraction and enzyme digestion of chromosomal proteins. Prestaining of silver nitrate or silver carbonate also blocked the fluorescences of protein dyes. However, removal of chromosomal DNA affected the silver-carbonate but not the silver-nitrate staining; the fluorescences of DNA dyes were blocked by the prestaining of silver carbonate but not silver nitrate. Chromosomal protein labelling was released only slightly and its relative amount between BrdU bifilarly substituted and unifilarly substituted chromatids was unchanged during pretreatment of Hoechst plus black light. We speculate that chromosomal non-histones are the targets for silver-nitrate stain, and DNA-non-histone complexes for silver-carbonate stain.