Using extract from alkanet (Alkanna tinctoria) as a source of both a red lipid stain and a blue counterstain for histology.

Alkanet (Alkanna tinctoria) is a plant native to and cultivated in parts of Europe, Asia and the Middle East. It has been used for thousands of years as a medicinal agent and as a colorant for textiles, food and cosmetics. An extract from the root of this plant has been used with a mordant to stain nuclei. We describe here the versatility of different extracts from this plant to stain lipids red and to counterstain certain other tissue elements blue. The color variation and selective differential staining is due to solvent polarity and pH. Extracts contain numerous chemical species, all of which are derivatives of the indicator dye, naphthazurin. Our red extract is nonionic below pH 7 and partitions from its somewhat polar solvent of 100% isopropanol to nonpolar lipids. The blue extract is dianionic at high pH in 70% isopropanol and binds ionically to cationic tissue structures such as collagen, muscle and cytoplasm of other cells.

Betacyanins are plant-based dyes with potential as histological stains.

Interest is increasing in certain parts of the world in replacing synthetic dyes with dyes from natural sources, particularly from plants. Although textile dyers have used various groups of natural dyes, microscopists generally have restricted their use to anthocyanins. Recently, however, another class of plant-based dyes has found some favor, the betacyanins. Betacyanins are a group of red and violet betalain dyes found only in certain plants of the order Caryophyalles and in Basidiomycetes mushrooms. Although the chemical structures of betacyanins are known, little use has been made of that information to understand or predict their behavior with biomedical specimens. We investigated two common, widely distributed betacyanin-containing plants, edible beets (Beta vulgaris) and wild pokeweed (Phytolacca americana). Aqueous alcoholic extracts were made from beet root and pokeweed berries, adjusted to pH 4.1 or 5.3 and used together with Harris' hematoxylin to stain histological sections. We used a methanolic extract of pokeweed berries, pH 3.0, to stain cultured mycological specimens. Both extracts produced satisfactory staining that was equivalent to that of eosin Y, although the colors were more muted with the beet root extract. Epithelial cytoplasm, muscle, collagen and erythrocytes were well demonstrated. Betanin is the predominant component of beet root extract; it possesses one delocalized positive charge and three carboxylic acid substituents. The dyes are weak acids and the carboxylate anions are more diffuse than for eosin Y; this produces weaker bonding to tissue cations. The principal colored component of pokeweed berries, prebetanin, possesses a sulfonic acid group as well as carboxylic acids, which favors acid dyeing and more intense coloration. Both dyes show potential for hydrogen bonding and to a much lesser extent for some types of van der Waals forces. Complex formation with metals such as aluminum to create a nuclear stain is not likely with beet root dyes nor is it possible with pokeweed dyes. Betacyanins are suitable for staining microscopy preparations in place of other red acid dyes such as eosin. Of the two dyes tested here, prebetanin from pokeweed berries was superior to betanin from red beet roots. These berries are widely distributed and readily collected; the extraction procedure is simple and does not require expensive solvents.

Anthocyanins from a single botanical source can be used as a replacement for hemalum and eosin.

For various reasons, histologists in several parts of the world have tried to replace hematoxylin and eosin with locally available plant dyes of the anthocyanin family. Blue or violet nuclear stains have been created by combining an anthocyanin with iron or aluminum ions at low pH. Obtaining a pink or red cytoplasmic counterstain, however, has not been achieved previously, even with a red solution of anthocyanin, because the chemistry of the colorant does not allow bonding to cytoplasmic materials and collagen. We used two extracts from the petals of common mallow, Malva sylvestris, to create both a blue nuclear stain and a red counterstain. The two extracts contained two chemically distinct types of anthocyanins. The first extract contains vic-hydroxyls capable of complexing aluminum ions; its flavylium core is cationic. The second type lacks vic-hydroxyls on its core structure, but includes pendant glucosides that contain a malonic acid ester with a free carboxyl substituent. The precise identity of the first anthocyanin currently is unknown, but likely is one or more of the common anthocyanins such as cyanidin, delphinidin or petunidin, which complex readily with aluminum. The second anthocyanin is malonated malvidin, which does not complex with aluminum, but is anionic at the pH used here. The overall visual effect of applying the two anthocyanin extracts is remarkably similar to that of hemalum plus eosin.

Molecular stabilization and complexation: the secrets of making a nuclear-selective histological stain from naturally occurring anthocyanins without oxidation.

The natural colorant, roselle, found in Hibiscus sabdariffa, has been used as a histological dye since at least 1976. As a simple extract roselle acts as a general red counterstain, but when treated with an oxidant and metallic mordant it functions as a useful blue nuclear-selective stain. In the past 40 years it has been assumed that oxidation is necessary when preparing a stain from roselle, as of course it is for the related flavonoid dyes hematoxylin and brazilin. However, the chemistry of roselle argues against this. Roselle is a mixture of four closely related compounds: delphinidin 3-sambubioside and cyanidin 3-sambubioside, delphinidin 3-glucoside and cyanidin 3-glucoside. Each of these in turn can exist in 8 different configurations in a complex state of equilibrium largely dependent upon pH. Of these compounds, only three are colored. In plants, and in their complex extracts, formation of these colorless compounds is inhibited by the presence of other colorless aromatic molecules, a stabilizing process termed co-pigmentation. The color of such extracts may be red in the acid range and blue or violet above neutrality. Complexation (chelation) with metal cations occurs above pH 2.8 or 3.0, depending upon the concentration of the metal. Prior oxidation is not needed for such chelation to occur. With Al3+ the resulting metal complex, which we term rosalum, is violet or blue. Following spectrophotometric investigation of this complexation, we were able to formulate a nuclear-selective violet/blue stain without the use of an oxidant.

Use of roselle extracted from Hibiscus sabdariffa for histological staining: a critical review and rational stain formulation.

Roselle is the common name for a mixture of anthocyanin dyes derived from the plant, Hibiscus sabdariffa. During the past two decades, a sizable, but conflicting, body of literature has supported the use of roselle as a biological stain, and more specifically as a substitute for hematoxylin for staining nuclei selectively. We review the literature and suggest a rational explanation for divergent findings. When used without oxidation and mordanting, roselle is an indiscriminate oversight stain. With appropriate oxidation and mordanting, roselle can be an effective nuclear stain. We propose here use of a stain that is formulated rationally and followed by treatment in a differentiating solution that also acts as a counterstain. We also offer suggestions for improving roselle for the general scientific community.