5. Artefacts due to poor mounting (imperfect dehydration and clearing, cloudiness, milkiness or opacity of section; folds; wrinkles; turned-over edges; tears in section caused by striking it with balsam-dropper or needle; air-bubbles; lack of balsam).
6. Dirt and foreign-material (opaque and translucent dirt, above or below section; coloring-matter in balsam; ink; pigment from pencil; cotton-, silk, wool-, linen-, vegetable and paper-fibres, hairs, desquamated squamous epithelium, portions of insects, etc.)
CHAPTER XXV.
STAINS AND STAINING METHODS.—NUCLEAR AND PROTOPLASMIC STAINS.
THEORIES OF STAINING. The exact nature of the process of staining has not yet been determined. Various theories have been advanced, explaining the affinity of the tissues for certain dyes, on the ground of a chemical, mechanical or chemicophysical action. The chemical theory assumes the formation of an insoluble compound through the chemical combination of tissue and stain; the physical theory is based upon the assumption that the process is purely physical or mechanical, while the chemicophysical theory holds that it is neither purely physical nor purely chemical. The process is not controlled by the molecular weights alone of the substances concerned, but does depend upon the conditions controlling the formation of solutions in general. Therefore, the theory most widely accepted at the present time is the solution-theory, which assumes that the staining-process is a solution of the dye in the tissue, and that the stained tissue-element is a fixed solution of the stain in its substance. This solution of the dye in the tissues may be a direct action between the two (direct or substantive stains); or it may be brought about only by the interaction of a third substance (indirect or adjective stains). The third substance is called a mordant, and the combination of the dye with the mordant is known as a lake. The mordant may be added to the stain or to the tissue, either before or at the time of staining. Many of the fixing-fluids are mordants, particularly those containing chromic acid or its salts. Alum, iron, and many of the metals are the most commonly used mordants. In a general way acid mordants are used for basic colors, and basic mordants with acid colors.
The stains most commonly used in pathologic work are:—
1. Natural Dyes:—Hæmatoxylin and Carmine.
2. Aniline Dyes:—a, Acid.—Eosin, erythrosin, acid fuchsin, orange G, picric acid, sudan III, and scarlet R (Fett-ponceau).
b, Basic.—Methylene blue, methylene violet, thionin, toluidin blue, kresyl-echt-violett, methyl violet, gentian violet, crystal violet, basic fuchsin, dahlia, aniline blue, methyl green, iodine green, safranin, Bismarck brown, and vesuvin.
In a general way it may be said that basic stains are nuclear stains, and acid stains are protoplasmic. Neutral stains are usually diffuse stains; when formed by the combination of acid and basic dyes they usually act as selective stains for some especial tissue-element or cell-constituent. Metachromasia, in its narrowest sense, is the term applied to that staining-phenomenon, in which a single-chemical entity gives different colors to different tissue-elements. In this sense iodine, in its action upon glycogen and amyloid, is a true metachromatic substance. The majority of the so-called metachromatic stains, however, do not possess true metachromasia, since their metachromatic powers are dependent upon a mixture of two or more dye-stuffs in the one compound. The most important stains of this class are gentian violet, methyl violet, crystal violet, dahlia, thionin, toluidin-blue, polychrome methylene blue, methylene azure, and kresyl-echt-violett. The chief chromotropic substances are amyloid, mucin, mast-cell granules and cartilage. Metachromatic reactions are at their best usually when examined in water; they are affected by alcohol and usually destroyed by carbol-xylol. Sections stained by metachromatic stains should be quickly dehydrated by absolute alcohol and blotting, and cleared in xylol, when mounted in balsam.
I. NUCLEAR STAINS.