γ. Staining of the dry specimen.
Staining methods may be classified according to the purpose to which they are adapted.
We use first those which are suitable for a simple general view. For this it is sufficient to use such solutions as stain hæmoglobin and nuclei simultaneously. (Hæmatoxylin-eosin, hæmatoxylin-orange).
Occasionally a stain is desirable which only brings out, but in a characteristic manner, a special kind of cell, e.g. the eosinophils, mast cells, or bacteria. Single staining is attained on the principle of maximal decoloration. (Cp. E. Westphal.)
Finally, we have panoptic staining; that is, by methods which bring out, as characteristically as possible, the greatest number of elements. Although we must use high magnifications with these stains, we are compensated by a knowledge of the blood condition that cannot be reached in any other way. A double stain is generally insufficient, and at least three different dyes are used.
Successive staining was formerly used for this purpose. But everyone who has used this method knows how difficult it is to get constant results, however careful one may be in the concentration and time of action of the stain.
Simultaneous staining offers undoubted and important advantages. As there is much obscurity with regard to the principle on which it rests we may here shortly explain the theory of simultaneous staining.
We will begin with the simplest example: the use of picro-carmine, a mixture of neutral ammonium carmine and ammonium picrate. In a tissue rich in protoplasm, carmine alone stains diffusely, though the nuclei are clearly brought out. But if we add an equally concentrated solution of ammonium picrate, the staining gains extraordinarily in distinctness, in as much as now certain parts are pure yellow, others pure red. The best known example is the staining of muscle with picro-carmine, by which the muscle substance appears pure yellow, the nuclei pure red. If, however, instead of ammonium picrate we add another nitro dye which contains more nitro-groups than picric acid, for example the ammonium salt of hexa-nitro-diphenylamine, the carmine stain is completely abolished, all parts stain in the pure aurantia colour. The explanation of this phenomenon is obvious. Myosin has a greater affinity for ammonium picrate than for the carmine salt, and therefore in a mixture of the two combines with the yellow dye. Owing to this combination it is not now in a condition to chemically fix even carmine. Further, the nuclei have a great affinity for the carmine, and therefore stain pure red in this process. If, however, nitro dyes be added to the carmine solution, which have an affinity for all tissues, and also for the nuclei, the sphere of action of the carmine becomes continually smaller, and finally by the addition of the most powerful nitro body, the hexa-nitro compound, is completely abolished. Connective tissue and bone substance, however, behave differently with the picro-carmine mixture, in as much as here the diffuse stain depends exclusively on the concentration of the carmine, and is quite uninfluenced by the addition of a chemical antidote. This staining can only be limited by dilution, but not by the addition of opposed dyes. We must look upon the latter kind of tissue stain not as a chemical combination, but as a mechanical attraction of the stain on the part of the tissue. We may also say: chemical stains are to be recognised by the fact that they react to chemical antidotes; mechanical stains to physical influences; of course always assuming, that purely neutral solutions are employed, and that all additions, which alter the chemical relation of the tissues such as alkalis and acids, or which raise or limit the affinity of the dye for the tissues, are avoided. A further consequence of this view is, that all successive double staining may be serviceably replaced by simultaneous multiple staining, if the chemical nature of the staining process is settled. In contradistinction, in all double stains, which can only be effected by successive staining, mechanical factors are concerned.
In the staining of the dry blood specimen, purely chemical staining processes are concerned, and therefore the polychromatic combination stain is possible in all cases.
The following combinations are possible for the blood: