A Nissl Method Using Buffered Solutions of Thionin

Experiments were performed in an attempt to obtain a rapid method for staining the chromophilic substance of formalin-fixed nerve cells differentially without resorting to over-staining and subsequent acid-decolorizing. A satisfactory procedure using thionin in dilute buffered solutions was developed: Prepare a stock solution of the dye using 1 g. in 100 ml. of distilled water. Prepare veronal-acetate buffers (Michaelis, 1931) in the range of pH 5 to pH 3.S. To each 10 ml. of the buffer add 0.5 ml. of the stock dye solution. After rinsing in CO₂-free distilled water place mounted or unmounted sections in this solution. (Freshly fixed material, 10μ to 20μ thick, is completely stained in 10 to 20 minutes but over-staining does not occur when longer times are allowed.) Return sections to distilled water (2 changes) and wash until diffusion of excess dye is no longer visible. Wash in 70% ethyl alcohol (2 changes) until diffusion of excess dye is no longer visible. Dehydrate in 95% ethyl alcohol and normal butyl alcohol, clear and mount.

Optimum staining of chromophilic material occurs at pH 3.65. Glial processes are well stained at pH 4.6. Nissl bodies and glial cytoplasm are purplish blue, nuclear chromatin is blue, background is clear at pH 3.65 but pale blue at pH 4.9.     

An Evaluation of Cresyl Echt Violet Acetate as a Nissl Stain

The new cresyl echt violet acetate, of which three different batches have been tested, proves to be a very useful Nissl stain. It is especially valuable for formalin-fixed, frozen-sectioned material. By using a buffered staining bath and controlled timing in dehydration it is possible, on paraffin embedded material, to use these dyes as progressive stains apparently specific for nucleic acids. With a saturated aqueous solution of the dye, especially when a mordant of lithium carbonate is used, it is possible to stain material that has been preserved in formalin for several years and also material from which nucleic acids have been removed. The dye is useful also for staining celloidin embedded material. With the buffered stain proposed, differentiation is much easier than with older methods which included a gross overstaining and a long destaining procedure.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

A Thionin Stain for Visualizing Bone Cells, Mineralizing Fronts and Cement Lines in Undecalcified Bone Sections

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

A Rapid Bulk Nissl Method

A simplified and time-saving Nissl method is described in which the fresh nervous tissue is placed directly into: dioxane, 65-70 ml.; water, 15 ml.; 95% alcohol, 20-15 ml.; and toluidine blue, 1 g. This solution fixes, stains, partially clears, and dehydrates in one procedure. The material is then sectioned according to a dry-ice method and mounted on the slide either with or without the use of the rapid albumen procedure. Thus it is possible to terminate an experiment one day and have the tissue ready for microscopic observation on the next.     

The Peracetic-Orcein-Halmi Stain: A Stain for Connective Tissues

A selective stain useful for the study of connective tissues is described. The stain demonstrates elastic and oxytalan fibers as well as fibrils in mucous connective tissues previously undescribed. Reticular fibers are not stained. The stain may be used on sections that have been fresh frozen or fixed in formalin or ethanol. Sections are deparaffinized, washed in absolute ethanol, oxidized in peracetic acid 30 min, washed in running water, stained in Taenzer-Unna orcein 15 min, 37°C, differentiated in 70% ethanol, washed in running water, stained in Lillie-Mayer alum hematoxylin 4 min, blued in running water, and counterstained 20 sec in a modified Halmi mixture of 100 ml distilled water, 0.2 gm light green SF, 1.0 gm orange G, 0.5 gm phosphotungstic acid and 1.0 ml glacial acetic acid. Sections are rinsed briefly in 0.2% acetic acid in 95% ethanol, dehydrated and mounted.     

Rapid Fluorescent-Antibody Stain Technique with Group A Streptococci

A rapid fluorescent-antibody stain technique used with young broth cultures of trypsinized group A streptococci is described. Results from 1,214 trypsinized cultures of group A streptococci employing the rapid stain method were equivalent, both in specificity and sensitivity, when compared to results obtained from identical non-trypsinized cultures stained with the longer standard technique of Cherry, Goldman, and Carski. Moreover, the rapid method requires less than 2 min to complete.     

Stain Technology: A Combined Elastic, Fibrin and Collagen Stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified VerhoefPs elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by Hssamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsia, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

A Technique for C-Banding Plant Chromosomes with Wright Stain

A technique is presented for C-banding plant chromosomes with a modified Wright stain. This procedure consistently produces brightly stained, well defined telomeric and interstitial heterochromatic bands, identifiable centromeric constrictions, and lightly stained euchromatic areas on chromosomes of rye.     

A One-Solution Stain for Spermatozoa

Fresh semen is allowed to liquefy 30-60 minutes and thin, even smears of it made on clean slides or cover glasses. The smears are fixed 3 minutes with an equal-parts mixture of alcohol and ether, then air dried. They are stained 5-7 minutes in an aqueous solution made by mixing 2 volumes of 5% aniline blue (water soluble), 1 volume of 5% eosin B and 1 volume of 1% phenol. Staining at 40-60°C. is recommended. After staining, the smears are washed with distilled water, air dried and mounted in balsam or synthetic resin. The method was used on over 2000 samples of dog semen and some human specimens. Good preservation and differentiation of cytological structures was obtained uniformly, but tests were not made with other species.     

Gallocyanin as A Nuclear Stain

Gallocyanin has been used successfully as a nuclear stain. Sections are cut by the freezing method of either fixed or unfixed tissue. The tissues are warmed (not exceeding 70°C.) for 2-4 minutes in the gallocyanin solution. A counterstain may be used if desired. The most effective are Biebrich scarlet, phloxine, or eosin Y. The sections are then dehydrated and mounted in clarite. The nuclear pattern is clearly demonstrated and the sections are permanent.     

A Quick-Mixed Aluminum Hematoxylin Stain

Two stock solutions are composed as follows: A) aluminum sulfate, sodium iodate and acetic acid in aqueous propylene glycol and B) hematoxylin in pure propylene glycol. When combined in specified proportions the stock solutions yield aluminum-hematein dissolved in nontoxic propylene glycol. The ready-to-use stain, prepared in small volumes as needed, performs well in paraffin sections of plant tissues.     

Wright's as A Differential Spore Stain

A method of differential spore staining utilizing Wright's stain diluted one to five in a phosphate buffer solution of pH 7.6 and following the general technic of the Dorner method is outlined. Spores are stained a deep blue while the cytoplasm of the sporangium is stained a pinkish red.     

A Bacterial Cell Wall Stain1

A new method is given to stain bacterial cell walls, especially of Escherichia coli and Bacillus cereus. The cells are smeared in water on a slide and, as soon as air-dry, are stained 3-4 minutes with a 1 % aqueous solution of new fuchsin. The smear is washed with water until the stain ceases to run and is then allowed to air dry. The slide is placed on a 50°C. warm plate for 10-20 seconds, and the smear is then covered with a thin film of a 1-2% solution of Congo red at a pH of about 9.5. The smear is ready for observation as soon as dry or it may be washed with water if desired before observation.     

A One Step Esterase Stain for Use with a Hemacytometer

When harvesting cells for macrophage cell cultures, our laboratory sought a method which would simultaneously confirm cell identity and allow enumeration of these cells. Such a procedure would reduce the time needed to assess the readiness of the cells for culturing (measuring their viability, and indicating their identity as well as their number).     

A Differential Stain for Nucleic Acids

An easily used trichrome stain consisting of orange G, methyl green, and toluidine blue is proposed as a method of differentiating desoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in cells. Carnoy's acetic-alcohol is the fixative of choice, though cold acetone is also satisfactory. Photomicrographs taken with ultraviolet and visible light show that the structures containing nucleic acid are exactly those which stain with methyl green and toluidine blue. Studies with nucleases and extraction of nucleic acids with cold and hot perchloric acid further indicate a specificityy of the dyes for DNA and RNA. Present experiments are directed toward using the stain for quantitative estimation of the nucleic acids.