Picro-Lithium Carmine.—The following is generally preferred for use—Lithium carmine solution, 100 cc.; saturated solution of picric acid, 270 cc.
There are several aniline dyes which are used for nuclear staining: methylene blue, methyl green, safranine, gentian violet, vesuvine, fuchsine, and Hoffmann’s blue.
The usual process is to stain in ¼ or ½ per cent. aqueous solutions and wash in methylated spirit. Methylene blue and methyl green have the reputation of being so readily washed out in the methylated spirit as to be worthless. This is obviated by washing the sections (when removed from the stain) in distilled water, previous to the differentiation in methylated spirit. Treated in this manner, the nuclear staining is very beautiful. This also applies to Hoffmann’s blue and partly to vesuvine; with the latter, however, it is not a necessity. Safranine and gentian violet worked better by transferring the sections directly from the stain into 90 per cent. alcohol.
Contrast Stains.—Very frequently other stains are used to dye the ground a colour which is in contrast to that employed for the nuclei. Brown, orange, or pink are used after nuclear blue or green. Carmine is generally counterstained yellow or indigo-blue; and fuchsine red, as in tubercle bacilli, is counterstained with nuclear blue. It is important that the ground stain should be made weaker than the principal stain, so that the whole tissue may be shown without detracting from the nuclei. The following colours are used as counterstains for animal sections, but they prove less useful for vegetable sections: benzo-purpurine, eosin, erythrosine, orange, acid rubin, and picric acid.
Examples of specific stains are fuchsine, methylene blue, and gentian violet for bacteria; osmic acid for fatty elements; victoria blue and rose bengale, for demonstrating elastic tissue; methyl violet, iodine, and safranine, for amyloid degeneration. Methylene blue is one of the most useful of aniline dyes, and one of the most variable in composition.
Iodine green, or methyl green, has long been in use as a reagent for amyloid, starchy matters, in ignorance of the fact that the reaction is due to the methyl violet, contained as an impurity in the iodine green. It is exceedingly difficult to obtain a green quite free from violet. As nuclear stains they are identical, and the amyloid reaction, being dependent wholly upon the contained violet, varies, not with the formula of the green, but with the extent to which it has been purified.
Cellulose reactions.—After the nuclear stains, the most important reagents to the botanist are those which affect cellulose and its several modifications. Pure cellulose is coloured yellow by iodine, the colour being changed to a blue on the addition of slightly dilute sulphuric acid, or a strong solution of zinc. Solutions containing iodine, iodide of potassium, and chloride of zinc, give a violet reaction with unaltered cellulose, and yellow with lignine.
Schulze’s zinc re-agent must be used with a certain amount of caution, as the chloride of zinc and potassium undergo decomposition. The formula now in use is as follows: Take of zinc chloride solution (sp. gr. 1·85) 70 cc., potassium iodide 10 grammes, iodine 0·1 gramme; but this solution can only be employed as a re-agent and not as a dye, and structures stained with it cannot be mounted in any of the ordinary media, and the only fluid for ringing them down is caoutchouc cement.
Cellulose can be stained permanently by carmine, hæmatoxylin, nigrosine, methylene blue, safranine, and fuchsine. The aniline dyes are used in dilute aqueous solutions containing one-eighth or one-fourth per cent. of dye. When the cellulose undergoes the change known as lignification its reactions are altered. It is coloured yellow by chloro-zinc iodine, red by phloroglucin, yellow by aniline chloride. The two latter are much assisted by hydrochloric acid. The results of these reactions also cannot be preserved in the usual mounting media.
Sections containing mixed tissue, partly unaltered cellulose and partly lignified, give striking results with aniline dyes, and with this additional advantage can be preserved for years.