Kiyoshi Harada

The Histochemical Significance of Staining Polysaccharide Sulfate Esters with Gentian Violet

The technic recommended is: Fix in any fixative, dehydrate in alcohol, embed, and cut paraffin sections. Stain 24 hr or overnight in 1:250,000 or 1:1,280,000 gentian violet, blot with filter paper, decolorize 5 min in an aniline-xylene mixture, clear in xylene, and cover in balsam. Results: Polysaccharide sulfate ester and heparin are stained but hyaluronic acid is not. Polysaccharide sulfate ester and heparin are resistant to hyaluronidase. The differentiating action of the method is based on the strong basophilia of polysaccharide sulfate ester and the staining obtained is specific.

Fixative-Stain Sequences for Selective Demonstration of Juxtaglomerular Cells

The effects of 31 fixatives, containing alcohol, acids, formalin and metallic salts, and representing many of the standard fixatives, were observed for selectivity and intensity of staining of juxtaglomerular granules in mouse kidney. Four staining methods: 1:400,000 aqueous methyl violet 2B; Bowies ethyl violet-Biebrich scarlet; 1:200,000 aldehyde fuchsin; and periodic acid-Schiff were used. Fixatives containing HgCl2, trichloroacetic acid or formalin were found to be the most satisfactory for subsequent staining of the granules.

Selective and Contrast Staining of Granules in the Juxtaglomerular Complex

A review of four methods for staining juxtaglomerular cells revealed that one method may be highly selective for juxtaglomerular granules (JGG) whereas another may stain general cytological features in addition to the granules. The kind of research undertaken would determine the particular method to be used. Haradas (1952) method, which uses a 1:400,000 solution of gentian violet is recommended as the highly selective stain, and the Masson-Goldner stain after a Ciaccio type fixation is best for cytological detail combined with clear tinctorial contrast of the JGG.

The Nature of Mycobacterial Acid-Fastness

Phenol is not essential to acid-fast staining, for it will occur in the absence of phenol where such lipoid-soluble basic dyes as night blue, Victoria blue B or Victoria R are used; it is essential for acid-fast staining with water soluble basic dyes such as basic fuchsin. When phenol is added to the staining solution, such water soluble basic dyes behave in effect like their lipid-soluble counterparts. The loss of mycobacterial acid-fastness with carbol-fuchsin after bromination or chromation indicates that this phenomenon is related to the presence of unsaturated lipids in the bacterial cells. Within the cells these acid-fast lipids are bound in such a way that they are easily removed from all mycobacteria by hot dilute HCl; from leprosy bacilli alone they are easily removed with hot pyridine. From the results of various blocking reactions it appears that carboxyl and especially hydroxyl groups of these cellular lipids are essential to the acid-fast reaction of mycobacteria.

Rapid demonstration of juxtaglomerular granules with alcoholic crystal violet.

Adult mouse kidney fixed in 9 commonly used fixatives was embedded in paraffin, sections prepared as usual, and tests made with 16 different dyes (mostly triphenyl methanes) for efficacy in staining of juxtaglomerular granules. On the basis of the tests, staining of dewaxed and hydrated sections in 0.5% crystal violet in 70% ethanol for 3 min, rinsing in tap water, blotting and completion of dehydration in a 1:1 aniline-xylene mixture, clearing in xylene, and covering in a synthetic resin, is recommended. Tissues fixed in dichromate-containing fixatives required a prestaining oxidation with acidified KMnO4, but this was optional for other fixatives. In addition to crystal violet: ethyl violet, new fuchsin, and victoria blue 4R were also found to be effective stains for the granules.

Effect of Prior Oxidation on the Acid-Fastness of Mycobacteria

Oxidants which attack ethylenes to form aldehydes were effective in increasing the acid-fastness of mycobacteria. Potassium permanganate, performic acid, and peracetic acid were found to be effective. With the permanganate Ziehl-Neelsen stain, acid-fast bacilli seemed to be more strongly stained and more numerous than they did with the unmodified Ziehl-Neelsen method. Moreover, as a practical advantage, the bacilli could be discerned at lower magnification.

Selective staining of mast cell granules with chrysoidin.

A simple chrysoidin stain (0.5% aqueous solution, 5-10 min) is selective for mast cell granules; alum-hematoxylin-chrysoidin stains histological features in addition to the granules and periodic acid-Schiff can be used for cytological and histochemical features. Selective chrysoidin staining is probably due to strong basophilia of mast cell granules.

An Improved Crystal Violet Method with Alkaline Differentiation for Juxtaglomerular Granules

In the previous alkaline crystal violet method for selectively demonstrating juxtaglomerular (JG) granules (Harada 1971), the staining solution was found to be unstable. Subsequent testing has shown that the alkali is equally effective if applied after a nonalkalized aqueous solution of crystal violet has been applied for the staining, thus allowing stable stock solutions of the staining reagents to be used. The new procedure is as follows:Sections of 4 μ thickness from adult mouse kidney fixed in phosphate-buffered 10% formalin were cut from paraffin-embedded material and attached to slides with albumen adhesive. They were deparaffinized, hydrated, and washed in tap water.

GRAM DIFFERENTIATION WITHOUT THE USE OF IODINE

A difference in the decolorization of the two types of cells is essential for Gram differentiation; Gram-positive cells being less decolorizable than Gram-negative cells. In practice, if a high molecular weight alcohol or aniline is used, a successful differentiation of both cells can be made by Gram procedures without iodine. Iodine plays a secondary role in that by its application, all cells are made less decolorizable than they would be without iodine. The formation of a dye-iodine precipitate within the cells seems to be the best explanation of the experimental findings.

Staining Juxtaglomerular Granules with Basic Fluorescent Stains

Many basic fluorescent dyes stain juxtaglomerular granules to produce characteristic colors in ultraviolet light. The stain is applied to paraffin sections of tissues fixed in 2% calcium acetate-10% formalin or in phosphate-buffered 10% formalin. Procedure: Bring section to water, stain 0.5 min in Delafield hematoxylin, wash in tap water, stain 3 min in a 0.1% aqueous solution of basic fluorescent dye (auramine O, acriflavine, acridine orange, coriphosphine O, acridine yellow, phosphine E, thioflavine T, berberine sulfate, atebrine or rivanol) and differentiate 1 min in 0.1% acetate acid (or omit this step). After washing in tap water, air dry with or without subsequent mounting in a resin. Juxtaglomerular granules stain bright fluorescent yellow or orange against a dark background.