Selective Silver Impregnation of Degenerating Axons In The Central Nervous System

Rat and rabbit brains containing surgical lesions of 5-10 days' duration were fixed in 10% formalin (neutralized with calcium carbonate) for 1 week to 6 months. Frozen sections (15-20 n) were rinsed and then soaked 7 minutes in a 1.7% solution of strong ammonia in distilled water. Subsequent treatment was as follows: rinse; 0.05% aqueous potassium permanganate 5-15 minutes; 0.5% aqueous potassium metabisulfite, 2 changes of 2.5 minutes each; wash thoroughly in 3 changes distilled water; 1.5% aqueous silver nitrate, 0.5-1.0 hr.; 1% citric acid, 5-10 sec.; 2 changes distilled water; 1% sodium thiosulfate, 30 see.; 3 changes distilled water. Each section is then processed separately. Ammoniacal silver solution (450 mg. silver nitrate in 10 ml. distilled water; add 5 ml. ethanol; let cool to room temperature; add 1 ml. strong ammonia water and 0.9 ml. of 2.5% aqueous sodium hydroxide), 0.5-1.0 min. with gentle agitation. Reduction of about 1 minute is accomplished in: distilled water, 45 ml.; ethanol, 5 ml.; 10% formalin, 1.5 ml.; 1% citric acid, 1.5 ml. Rinsing; 1% sodium thiosulfate, 10 sec.; thorough washing followed by dehydration through graded alcohol and 3 changes of xylene or toluene complete the staining process. Normal nerve fibers are slightly stained to unstained, degenerating fibers, black. The treatment in potassium permanganate is critical since too little favors overstaining of normal fibers and too much abolishes staining of degenerating fibers.     

Silver Impregnation of Ciliated Protozoa by the Chatton-Lwoff Technic

The ciliates are manipulated at all stages by means of micropipettes and fine needles, proceeding by the following steps: Fix the material in a small receptacle with Champy's fluid 1-3 minutes following with Da Fano's solution for several hours. Transfer the specimens to a slide, withdraw excess fluid and embed them in warm (35°-45°C.) gelatin containing 0.05% sodium chloride. Refrigerate in a moist chamber until the gelatin has set, and then immerse 10-20 minutes in 3% silver nitrate (aqueous) at 5-10°C. Wash with cold distilled water, submerge the preparation in cold water to a depth of several centimeters and expose to a strong light for 10-30 minutes. Silver is deposited on various pellicular structures which then appear black in the dehydrated and mounted specimens. Neatly revealed are the many longitudinal and transverse fibrils of the “silverline system”, basal granules of the cilia, bases of buccal ciliary organelles, contractile vacuole pores and the cytoproct. None of these structures, which today are considered to be of inestimable value in comparative morphological and taxonomical studies of ciliates in general, is so precisely made evident by any other technic known to the author.     

Silver Impregnation of Nerve Fibers in Teeth After Decalcification With Ethylenediaminetetraacetic ACID

The difficulties in impregnating bony tissues, which occur after decalcification with acids or electrolysis are avoided by decalcification with ethylenediaminetetraacetic acid at pH 8.2-8.5. The decalcification of adult human teeth which have been cut to a thickness of 2-5 mm takes 1-2 mo. If frozen sections of the decalcified teeth are impregnated 24 hr in 20% AgNo₃, rinsed through 6 changes of 20% neutralized (CaCO₃) formalin, blotted thoroughly with a cloth and placed in an ammoniated silver solution for 15-20 min, reliable impregnation of nerve fibers is obtained. The stock ammoniated silver solution is prepared by adding concentrated NH₄OH to 10-20 ml of 20% AgNO₃ until the precipitate formed by it is dissolved and then adding a few drops of the silver solution until the first permanent opalescence of the mixture is obtained. From this 2 ml are diluted directly before use with 6 ml of distilled water and 4 drops of concentrated NH₄OH added. The diluted stock solution should be used for few (5-10) sections only. The rest of the technic is done in the routine manner.     

Triple Silver Impregnation for Selective Staining of Avian Nerves

Frozen sections of avian tissue fixed 7 days or longer in 10% formalin or formol-saline are cut at 20-50 μ, left in distilled water for 2 hr, and placed in 0.002% aqueous AgNO₃ for 3-4 days. Subsequent procedure is essentially that of Weddell and Glees. Sections are placed in 20% AgNO₃ for 30 min, then carried through 3 baths of 3% formalin in less than 10 min. Immediately thereafter they are washed 1-2 sec in a 0.1% solution of NH₄OH (cone) and placed in the ammoniacal silver solution (made with 20% AgNO₃) until the nerves become distinct, as seen under a microscope; usually, in about 15 min. After washing briefly, the sections are fixed in 5% Na₂S₂O₃ for 3-10 min, dehydrated, cleared, and mounted in the usual way.     

Formalin-Chloride Fixation to Improve Silver Impregnation of the Golgi Apparatus

The addition of a 1% concentration of alkaline earth or heavy metal chloride to a 15% neutral formalin solution was found to be much superior to a similar addition of nitrate. Of the 19 salts tried, BaCl₂ and CoCl₂ generally gave the best results with the tissues of Pheretima posthuma, Pila globosa, Rana tigrina, Cauia porcellus and Mus rattus. The CoCl₂ fixative was found to work more successfully with shorter time for fixation and subsequent impregnation by silver, than da Fano's cobalt nitrate technique. The results obtained with BaCl₂ fixative, were equally good and in some cases even excelled those obtained with Aoyama's CdCl₂. Fixatives containing MgCl₂ and ZnCl₂ were satisfactory but the results were not so good as were those obtained with barium and cobalt chloride fixatives. Fixatives containing nitrates generally required longer periods for impregnation and reduction, than those containing chlorides.     

Alcoholic Thionin Counterstaining Following Golgi Dichromate-Silver Impregnation

Brains of cats that had been fixed 2 months or longer in 10% formalin were cut into 3-6 mm. slices and impregnated by Golgi's dichromate-silver procedure (6% dichromate solution, 4-6 days; 1.5% silver nitrate solution 2 days). Sections 100 µ thick were cut after embedding in low melting point paraffin. Three changes of xylene and three of absolute alcohol were followed by staining 3-5 minutes in a saturated solution of thionin in absolute alcohol. The sections were dipped quickly in absolute alcohol and cleared in xylene, then differentiation was effected by an equal-parts mixture of absolute alcohol and xylene. A final clearing in three changes of xylene and mounting in Permount completed the process. Counter-staining was most successful when applied to freshly cut sections.     

Enhanced Reliability in Silver Impregnation of Terminal Axonal Degeneration-Original Nauta Method

Brains of rat with surgical lesions 3-5 days old are fixed in 10% neutralized formalin (excess of CaCO₃), 20 μ serial frozen sections cut therefrom and kept in neutralized formalin for an additional 24-48 hr. The sections are soaked in distilled water 12-24 hr, transferred to 50% alcohol containing 0.75 ml of concentrated NH₄OH (sp. gr. 0.91) per 100 ml 12-24 hr, placed in distilled water 2-3 hr and then in silver-pyridine solution (AgNO₃ 3% aq., 20 ml; pyridine, 1 ml) for 48 hr. Test sections are transferred directly to each one of 3 ammoniated silver-solutions, pH 12.8, 13.0 and 13.2, made as follows: To 200 ml of solution 1 (silver nitrate, 6.4 gm; alcohol 96%, 220 ml; NH₄OH (sp. gr. 0.91), 28 ml and distilled water, 440 ml) is added respectively 8-12 ml, 12-16 ml and 16-20 ml of solution 2 (2% NaOH) to give the pH desired. The test sections are studied and the optimal ammoniated silver solution chosen. Two baths of ammoniated silver are used, the section placed with continuous agitation into the first bath for 30 sec and the second bath for 60 sec. The sections are then transferred directly into a reducing bath (formalin 10%, 2ml; alcohol 96%, 5 ml; citric acid 1%, 1.5 ml and distilled water, 4.5 ml) for 2 min and from there to 5% Na₂S₂O₃ for 1 min, rinsed in 3 changes of distilled water, dehydrated and mounted.     

Suppression of Connective Tissue Impregnation in a Silver Technique for Demonstrating Nerve Fibers

A tissue pretreatment technique is introduced which effectively suppresses the silver impregnation of connective tissue and nompecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcobol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginnins with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilidng fresh hrozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation H suppressed by the use of mercuric chloride in the fixative and that the background supprgsion is related to the short fixation time with formol-sublimate.     

Golgi Impregnation After Formalin Fixation

Formalin fixed (10% aqueous) brain from cat, rabbit and man cut to blocks 3-4 mm. thick was placed in a mixture of potassium bichromate, 5 g.; chloral hydrate, 3 g. and water 90 ml. for 24 hours. The specimens were rinsed through 3 changes of water, and transferred through 3 changes of 1% silver nitrate, 1-3 minutes each, then placed for 24 hours in 1.5% silver nitrate. Frozen sections, 40-50 μ were dehydrated and mounted with a cover glass, using Permount. No deterioration of the stain was seen after 5 months. Some brains had been in formalin for 9 months; others only 7 days.     

Staining Paraffin Sections with Protargol 6. Impregnation and Differentiation of Nerve Fibers in Adrenal Glands of Mammals

A series of experiments with protargol staining of nerve fibers in mammalian adrenal glands has yielded the following procedure: Fix-1-2 days in a mixture of formamide (Eastman Kodak Company) 10 cc, chloral hydrate 5 g., and 50% ethyl alcohol 90 cc. Wash, dehydrate and embed in paraffin. Cut sections about 15 and mount on slides. Remove the paraffin and run down to distilled water. Mordant 1-2 days in a 1% aqueous solution of thallous (or lead) nitrate at 56-60°C. Wash thru several changes of distilled water and impregnate in 1% aqueous protargol (Winthrop Chemical Company) at 37-40°C. for 1 to 2 days. Rinse quickly in distilled water and differentiate 7-15 seconds in a 0.1% aqueous solution of oxalic acid. Rinse thru several changes of distilled water for a total time of 0.5 to 1.0 rain. Reduce 3-5 rain, in Bodian's reducer: hydroquinone 1 g., sodium sulfite 5 g., distilled water 100 cc. Wash in running water 3-5 min. and tone 5-10 min. in a 0.2% gold chloride solution. Wash 0.5 min. or more and reduce in a 2% oxalic acid solution to which has been added strong formalin, 1 cc. per 100. (Caution. This last reduction is critical and over-reduction can spoil an otherwise good stain; 15-30 seconds usually suffices, and the sections should show only the beginning of darkening to a purplish or gray color.) Wash, fix in hypo, wash, dehydrate and cover.     

Silver Impregnation of Degenerating Axons in the Central Nervous System: A Modified Technic

Frozen sections of formalin-fixed brains containing surgical lesions, were treated with 15% ethanol for 0.5 hr., soaked in 0.5% phosphomolybdic acid for 0.25-1.0 hr., and subsequently treated with 0.05% potassium permanganate for 4-10 min. (The duration of the latter treatment is critical and individually variable). Subsequent procedure is as follows: decolorize in a mixture of equal parts of 1% hydroquinone and 1% oxalic acid; wash thoroughly and soak sections in 1.5% silver nitrate for 20-30 min.; ammoniacal silver nitrate (silver nitrate 0.9 g., distilled water 20 ml., pure ethanol 10 ml., strong ammonia 1.8 ml., 2.5% sodium hydroxide 1.5 ml.) 0.5-1.0 min.; reduce in acidified formalin (distilled water 400 ml., pure ethanol 45 ml., 1% citric acid 13.5 ml., 10% formalin 13.5 ml.) 1 min.; wash, and pass section through 1 % sodium thiosulf ate (0.5-1.0 min.); wash thoroughly and pass sections through graded alcohols and xylene (3 changes); cover in neutral synthetic resin.     

Perfusion Method for Preparing Pig Brain Cortex for Golgi-Cox Impregnation

The perfusion procedure described in this paper produces high quality impregnation of pig visual and somatosensory cortical neurons with a Golgi-Cox solution. Starting within 30 min after death, pig heads were perfused with a fixative solution composed of a mixture (v/v) of liquid phenol, 5%; formalin, 14%; ethylene glycol, 25%; methanol, 28%; and water, 28% for two periods of 4 hr each. After perfusion, the heads were chilled for at least 18 hr. The entire brain was removed from the skull and then placed in 10% buffered formalin, where it remained for at least 10 days before taking the blocks that were to be immersed in the Golgi-Cox solution. Three weeks spent in the Golgi-Cox solution typically produced uniform neuron impregnation. The tissue blocks were then embedded in celloidin and sectioned at 120 μm. This procedure avoids the following difficulties: Golgi-Cox methods that produced excellent results with rodent or primate tissue were unsuccessful with pig tissue, placing fresh tissue in Golgi-Cox solution resulted in incomplete neuron impregnation, and immersion fixation in 10% buffered formalin without perfusion resulted in excessive staining of glia.     

Nitrogen assimilation and transport in carob plants

Most of the nitrate reductase activity (80%;) in carob ( Ceratonia siliqua L. cv. Mulata) is localised in the roots. The nitrate concentration in the leaves is relatively low compared to that in the roots, suggesting that nitrate influx into the leaf may be a major factor limiting the levels of nitrate reductase in the shoot. Transport of nitrate from root to shoot appears limited by the entrance of nitrate into the xylem. In order to study this problem, we determined the nitrate concentrations and nitrate reductase activities along the roots of nitrate-grown plants, as well as the composition of the xylem sap and the nitrate levels in the leaves. Some of the the bypocotyl, in order to bypass the loading of nitrate into the xylem of the roots. The results show that the loading of nitrate into the xylem is a limiting step.
The cation and anion concentrations of nitrate- and ammonium-fed plants were similar, showing almost no production of organic anions. In both nitrate- and ammonium-fed plants, the transport of nitrogen from root to shoot was in the form of organic nitrogen compounds. The nitrate reductase activity in the roots was more than sufficient to explain all the efflux of OH⁻ into the root medium of nitrate-fed plants. In carob plants the K-shuttle may thus be operative to a limited extent only, corresponding to between 11 and 27%; of the nitrate taken up. Potassium seems to be the cation accompanying stored nitrate in the roots of carob seedlings, since they accumulate nearly stoichiometric amounts of K⁺ and NO⁻₃.     

Hydroxyapatite-mediated transfer of DNA from diluted solutions to nitrocellulose or nylon membranes

Transfer of DNA (from 0.1 to 10 micrograms) from diluted solutions of variable volumes (1-10 ml) and various composition (2 M NaCl; 4 M LiCl, 8 M urea; 4 M CsCl; 20% sucrose) to nitrocellulose or nylon membranes was achieved with the use of hydroxyapatite. This absorbent that binds nucleic acids effectively and independently of ionic strength and composition of solution (except for chelators and phosphate ions) easily dissolves in small volumes of acids (for example, in 10% TCA). This phenomenon provides the opportunity to deliver the acid-insoluble precipitates to membrane filters. After alkaline denaturation on the filter followed by a fixation step (baking or UV irradiation for nitrocellulose or nylon filters, respectively), DNA hybridizes effectively with nick-translated DNA probes. The method is simple, reproducible, sensitive, and useful for working with diluted DNA solutions containing interfering substances.     

Formulation of trichloroacetic acid peeling solution: a bibliometric analysis

Since the beginning of this century, trichloroacetic acid solutions of various concentrations have been used for chemical exfoliation. These solutions have been prepared by using four different formulas. To prepare a 50% solution, for instance, water may be added to 50 g of trichloroacetic acid crystals until 100 ml of solution is obtained (weight-to-volume solution). Alternatively, 50 g of water may be added to 50 g of trichloroacetic acid crystals (weight-to-weight solution), or 50 g of trichloroacetic acid crystals may be solved in 100 ml of water (weight-plus-volume solution). Finally, a saturated trichloroacetic acid solution (or "100% solution") may be diluted by an equal volume of water (dilution). Depending on the method used, these so-called 50% solutions contain 40 to 71 weight-to-volume percentages of trichloroacetic acid. From a review of 120 publications on trichloroacetic acid peeling that have appeared since 1926, it was concluded that the authors of 87 of these publications (73 percent) did not report their formula for the trichloroacetic acid solution. Any one of the four methods was reported to have been used by the 33 authors who did report their formula. Eight of 10 internationally reputed pharmacopeias were found not to include the formula of a trichloroacetic acid solution. Proper evaluation of results and prevention of complications of trichloroacetic acid chemexfoliation is only feasible if both the concentration and the formula of trichloroacetic acid solution are reported by the author. Practitioners who use a trichloroacetic acid solution need to establish that the concentration of the solution they apply corresponds with that of the solution reported in the literature.     

Molecular approaches to the analysis of nitrate assimilation

Abstract. The application of molecular approaches such as mutant analysis and recombinant DNA technology, in conjunction with immunology, are set to revolutionize our understanding of the nitrate assimilation pathway. Mutant analysis has already led to the identification of genetic loci encoding a functional nitrate reduction step and is expected to lead ultimately to the identification of genes encoding nitrate uptake and nitrite reduction. Of particular significance would be identification of genes whose products contribute to regulatory networks controlling nitrogen metabolism. Recombinant DNA techniques are particularly powerful and have already allowed the molecular cloning of the genes encoding the apoprotein of nitrate reductase and nitrite reductase. These successes allow for the first lime the possibility to study directly the role of environmental factors such as type of nitrogen source (NO₃⁻ or NH₄⁺) available to the plant, light, temperature water potential and CO₂ and O₂ tensions on nitrate assimilation gene expression and its regulation at the molecular level. This is an important advance since our current understanding of the regulation of nitrate assimilation is based largely on changes of activity of the component steps. The availability of mutants, cloned genes, and gene transfer systems will permit attempts to manipulate the nitrate assimilation pathway.     

Staining of Nervous Tissue by Protein-Silver Mixtures

A staining method for nerves in paraffin sections is described in which an egg albumen-silver nitrate mixture is the impregnating solution. Blocks of tissue are fixed in Bouin's fixative, formol, Huber's fixative or formol-acetic-alcohol, and decalcified if necessary in Bensley's decalcifier. Sections are impregnated overnight, in the dark, at 37-56°C in a solution containing 50 ml of filtered, aqueous 0.5% dried egg albumen with 1.8-2.5 ml of 2% silver nitrate and adjusted to pH 8.2-8.3 by the addition of ammonia. The sections are then rinsed in distilled water and the silver reduced in a mixture of hydroquinone, 1 gm; anhydrous sodium sulfite, 10 gm and distilled water, 100 ml. The remainder of the process consists of washing, gold toning, fixing in 5% sodium thiosulfate, washing, dehydrating, clearing and mounting. Casein may be used as an alternative to egg albumen in the impregnating solution (0.5% casein, 50 ml; 2% silver nitrate, 1 ml). The pH value of the solution may be adjusted by a boric acid-borax buffer or ammonium hydrogen tetraborate in the place of ammonia.     

A Rapid Silver Impregnation Method for Nervous Tissue: A Modified Protargol-Peroxide Technic

A rapid, reliable silver impregnation method is described for nervous tissue fixed in formol-saline, Bouin or Sum. Sections are impregnated for 10-15 minutes at room temperature or 37 C in a solution containing 0.5 g Protargol-S, 0.005-0.01 g allantoin, 1 ml of 1% Cu[NO₂]₂, 1 ml of 1% AgNO₃. and 1-2 drops of 30% H₂O₂ in 100 ml distilled water. Thereafter the dons arc reduced in a hydroquinone-formalin solution. This is followed by gold toning and subsequent reduction and mounting. Alternatively. following the first reduction, the silver image can be intensified by placing sections in a silver-allantoin bath which is followed by reduction and mounting. This method is very reliable and selective, making it suitable for general routine and research use.     

Development of a miniaturized nitrate reduction test for the identification of oral bacteria

A miniaturized nitrate reduction test (MNRT) for oral bacteria was developed and its reliability compared with a conventional nitrate reduction test (CNRT). In the MNRT 100 μl aliquots of freshly grown heavy suspension of various oral bacterial species, in physiological saline, were added to equal volumes of 0.1% filter-sterilized KNO₃ solution in distilled water in wells of transparent plastic plates. Duplicate plates were incubated aerobically or anaerobically at 35°C for 12–15 h. At the end of the incubation period the test was performed by adding either a trace amount of a non-liquid reagent (mixture of l-(+)-tartaric acid, sulfanilic acid and 1-naphthylenediamine dihydrochloride, 10:1:1, wt/wt) or conventional liquid reagents A and B (sulfanilic acid and N,N-dimethyl-1-naphthylamine). In the conventional nitrate reduction test (CNRT), tubes of a basal anaerobic broth were inoculated with the same bacterial species used for MNRT, and the nitrate reduction tests performed after anaerobic incubation of the cultures for 4–6 days. Several hundred anaerobic and facultative bacterial isolates belonging to genera Veillonella, Bacteroides, Fusobacterium, Selenomonas, Actinomyces and Capnocytophaga were characterized by MNRT and CNRT. Analysis of the data showed that MNRT and CNRT systems were comparable. In the MNRT system Veillonella parvula and Selenomonas sputigena were capable of reducing nitrate only under anaerobic conditions. Actinomycetes reduced the nitrates under aerobic and anaerobic conditions, while all black-pigmented Bacteroides, Fusobacterium and Capnocytophaga species did not reduce nitrate. These findings suggest that the MNRT is reliable, rapid and may be conveniently used in clinical or research laboratories with a heavy microbiological work load.     

Epidermal and Cuticular Mounts of Plant Material Obtained by Maceration

Mounts of leaf and stem epidermises or bare cuticles, useful in both general anatomical and specialized phylogenetic studies, can be prepared by a maceration process using Jeffrey's solution (equal volumes of 10% aqueous CrO₃ and 10% HNO₃). Leaves, including those of conifers, and stems with cuticles thick enough to maintain integrity when isolated are amenable to this process. Dried specimens are hydrated by boiling in water; fluid-preserved specimens are washed thoroughly in water; fresh specimens need no pretreatment. Specimens are cut to a convenient mount size and trimmed so as to allow adequate and even penetration of the macerating fluid. Laminar leaf segments are left with one edge untrimmed so that upper and lower epidermises remain contiguous. Cylindrical leaf and stem segments are slit lengthwise through about half their thickness. Specimens are macerated in Jeffrey's solution for 1 to 4 days until unwanted tissues are loosened and easily freed from the epidermis (or bare cuticle, if that is desired). The macerating fluid is then washed out completely with changes of water and specimens are stained in a 0.5% aqueous solution of safranin. Dehydration is accomplished with several changes of tertiary butanol. All unwanted tissues not removed by agitation during previous steps of the process are removed by teasing prior to mounting. Specimens are then mounted on slides in Canada balsam and dried in the usual manner.