Improved Movat Pentachrome Stain

Movat in 1955 developed a staining method which demonstrates collagen fibers, mucin, muscle fibers, elastic fibers, fibrin and fibrinoid changes in a single section. His procedure was considered excellent by Lynch et al. (1969)     

Acceleration of the Movat Pentachrome One Stain by Heat

The conventional staining time for Movat's pentachrome I stain (Arch. Path., 60: 289-295, 1955) was shortened from about 18-19 hr to about 2.5 hr. The ammoniated alcohol and the resorcin-fuchsin staining baths were heated to 56 C. All other steps in the technic were performed at 25-27 C. Staining properties of paraffin sections of many types of tissue fixed in formalin, formol-sublimate-acetic, or in Bouin's fluid, showed that staining with resorcin-fuchsin at the elevated temperature gave the same results as staining at room temperature.     

Differential Staining of Calcified Tissues in Plastic Embedded Microtome Sections by A Modification of Movat's Pentachrome Stain

Movat's pentachrome I stain has been adapted and modified as a stain for Undecalcified bone sections. After embedding in methyl methacrylate, this procedure yields consistently good results, with an excellent and colorful contrast between mineralized and unmineralized compartments of both cartilage and bone. in addition, osteoblats, osteoclasts, and other cells and tissue components can easily be differentiated. the staining properties of the lacunar wall surrounding the osteocytes are considered to reflect various states of osteocytic activity. the method is especially useful for the study of bone growth and bone repair, and as a stain for conventional histomorphometry and computer-assisted image analysis in bone biopsies.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4′,6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

A Sensitive Myelin-Sheath Stain Particularly Useful for Small Mammals and Neonatal Cats

Staining of myelinated fibers including the delicate myelin sheaths of infantile animals is as follows: perfuse the anesthetized animal with a pH 7.4 posphate-buffered fixative, either 10% formalin, 6% gluteraldehyde or a mixture containing 3% gluteraldehyde and 2% acrolein. Dissect out the brain or spinal cord and continue fixation for at least 24 hr. Cut larger brains to 1 cm in at least one dimension. Wash in running tap water 2-3 hr and soak in 2.5% potassium dichromate in 1% acetic acid (the primary mordant) for 3-5 days in darkness. Wash at least 12 hr in running tap water. Dehydrate and embed in celloidin and store in 80% ethanol. Section at 25-60 μ into 80% ethanol. Wash 1-2 min in distilled water and then immerse in 1-2% ferric alum at 50 C for at least 1 hr (the secondary mordant). Wash in tap water and stain at least 1 hr at 50-60 C in 0.5% unripened hematoxylin in 1% acetic acid. Wash well in tap water and differentiate in a mixture containing 0.5% ferrityanide, 0.5% borax and 0.5% Na₂CO₃; 2 changes. Wash well in distilled water, then in tap water, and dehydrate, clear and mount. Myelin stains black, cell bodies stain tan, and the background is pale yellow. With minor modifications in timing, the method is applicable to frozen and to paraffin sections; the primary mordant being omitted in the freezing technique.     

A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.     

A New Pentachrome Method for the Simultaneous Staining of Collagen and Sulfated Mucopolysaccharides

Collagen is one of the most common fibers in the extracellular matrix, where sulfated mucopolysaccharides are also located. In addition, sulfated mucopolysaccharides are present in some globet cells and secretory glands. The objective of this article is to develop a new staining method that detects these two macromolecules simultaneously in the same sample. The method described stains tissues in five fundamental colors: collagen in red; sulfated mucopolysaccharides in violet; red blood cells in yellow; muscle in orange; and nuclei in green.As a conclusion, it will be interesting in the future to evaluate whether this method could be used as a basic histological method, as a histology teaching tool, or even in histopathological and cytopathological studies.     

A New Hematological Stain: I. Constituents and Methods of Use

The stain proposed by the author is quickly and simply prepared by mixing equal parts of the following permanent stock solutions:

The mixed stain is usable, for at least eight months, and is applicable to practically all hematological purposes: blood smears, fixed sections, frozen sections, and touch preparations. In the technics utilized to produce its action on preparations treated in different ways, the only variants are the methods for treating the cells, while the stain itself remains totally unchanged for all purposes. For blood smears fixed with methyl alcohol, the technic consists merely of pouring the stain on the slide, leaving it for 5 to 7 minutes, and then washing it off. On sections, a further process of differentiation with acid acetone is rapidly carried out. The various types of granules, including megakaryocyte and platelet granules, are clearly demonstrated. For frozen sections, the technic is extremely rapid, yet yields excellent differentiation.     

A Controllable Differential Stain for the Hypophysis by Adaptation of the Mallory Triple Stain

A selective and controllable staining method for the hypophysis has been developed with rat material, using Mallory's triple stain as a basis.

Fix in Zenker neutral formol for 6 hours. Longer fixation is undesirable. Transfer to 30% alcohol plus a few drops of a saturated solution of I₂ in aqueous KI over night. Gradually complete dehydration and clear in cedar oil. Infiltrate with a paraffin mixture (paraffin, rubber-paraffin, bayberry wax and beeswax). Section 3-Sμ. Hydrate to distilled water, placing a few drops of a KI-I₂ solution in the 50% alcohol. Stain in 1% acid fuchsia for 30 minutes. Rinse, and differentiate in a weak NH₄OH solution (one drop 28% NH₄OH to 200 cc. HOH). When differentiation is complete, transfer to a 0.5% phosphomolybdic acid solution for 3 minutes, after first stopping the differentiation with a 0.1% HC1 solution and then rinsing with distilled water. Stain for one hour in a solution of: 1% anilin blue, water soluble, 2% orange 6, and 1% phosphomolybdic acid. Rinse in distilled water plus a few cubic centimeters of the stain. Differentiate in 95% alcohol, transfer to absolute alcohol and clear in a mixture of 30% oil of cedar, 40% oil of thyme, 15% absolute alcohol and 15% xylene. Finally, transfer to xylene and mount.     

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.     

A Note on the Gram Stain

A modification of the Gram stain in which iodine-alcohol is substituted for 95% alcohol as a decolorizing agent has been found particularly useful in staining Gram-positive organisms in tissues and also for smears. The technic for tissue sections follows:

1. Apply nuclear stain.
2. Wash.
3. Stain in Hucker's gentian violet 2 to 3 minutes (i. e. 1 part Sat. Alc. Sol. crystal violet to 4 parts 1% Aqu. Sol. ammonium oxalte).
4. Wash in water.
5. Stain in Gram's iodine 5 minutes.
6. Wash in water.
7. Decolorize in 95% alcohol to which enough tincture of iodine has been added to give a mahogany color.
8. Counterstain.
9. Dehydrate and mount.

     

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.     

Colorimetry for the Stain Technologist. I. The Specification of Color

This paper describes color specification for the stain technologist. The principles of color stimulus specification are reviewed in terms of the conventions of the Commission Internationale de l'Eclairage (CIE). The text is largely self-contained and has been written so that it can be understood easily by a reader with no prior knowledge of color science. The paper starts with definitions of color and related psychological, psychophysical and colori-metric terms. X, Y, Z color space is described. It is shown that any color stimulus may be unambiguously defined in terms of a set of three numbers. The CIE 1931 Chromaticity Diagram is described. Worked examples are given for the calculation of tristimulus values and chromaticity coordinates using three different illuminants. The usefulness of color specification is illustrated by a number of examples using Romanowsky stained blood cells or Papanicolaou stained epithelial cells from the uterine cervix.     

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.