1. Combined staining with acid dyes. The best known example is the eosin-aurantia-nigrosin mixture, in which the hæmoglobin takes on an orange, the nuclei a black, and the acidophil granulations a red hue.
2. Mixtures of basic dyes. It is possible straight away to make mixtures consisting of two basic dyes. As specially suitable we must mention fuchsin, methyl green, methyl violet, methylene blue. On the other hand, mixtures of three bases are fairly difficult to prepare, and the quantitative relations of the constituents must be exactly observed. For such mixtures, fuchsin, bismarck brown, chrome green, may be used.
3. Neutral mixtures. These have played an important part in general histology, from the time that they were first introduced by Ehrlich into the histology of the blood up to the present day; and deserve before all others a full consideration.
Neutral staining rests on the fact, that nearly all basic dyes (i.e. salts of the dye bases, for instance, rosanilin acetate) form combinations with acid dyes (i.e. salts of the dye acids, for instance, ammonium picrate) which are to be regarded as neutral dyes, such as rosanilin picrate. Their employment offers considerable difficulties as they are very imperfectly soluble in water. A practical application of them was first possible after Ehrlich had ascertained that certain series of the neutral dyes are easily soluble in excess of the acid dye, and so the preparation of solutions of the required strength, readily kept, was made possible. Among the basic dyes which are suitable for this purpose are those particularly which contain the ammonium group, especially methyl green, methylene blue, amethyst violet[5] (tetraethylsafraninchloride), and to a certain extent pyronin and rhodamin also. In contradistinction to these, the members of the triphenylmethan series, such as fuchsin, methyl violet, bismarck brown, phosphin, indazine, are in general less suited for the purpose, with the exception of methyl green already mentioned. The acid dyes specially suited for the production of soluble neutral stains are the easily soluble salts of the polysulpho-acids. The salts of the carbonyl acids and other acid phenol dyes are but little suitable: and least of all, the nitro dyes. Specially to be mentioned among the acid dye series are those which can be used for the preparation of the neutral mixtures: orange g., acid fuchsin, narcëin (an easy soluble yellow dye, the sodium salt of sulphanilic acid—hydrazo-β-naphtholsulphonic acid).
If a solution of methyl green be allowed to fall drop by drop into a solution of an acid dye, for instance orange g., a coarse precipitate first results, which dissolves completely on the further addition of the orange. No more orange should be added than is necessary for complete solution. This is the type of a simple neutral staining fluid. Chemically the above-mentioned example may be thus explained; in this mixture all three basic groups of the methyl green are united with the acid dye, so that we have produced a triacid compound of methyl green.
Simple neutral mixtures, which have one constituent in common, may be combined together straight away. This is very important for triple staining, which can only be attained by mixing together two simple neutral mixtures, each consisting of two components. A chemical decomposition need not be feared. We thus get mixtures containing three and more colours. Theoretically there are two possibilities for such combinations:
1. Staining mixtures of 1 acid and 2 basic dyes,
| e.g. orange—amethyst—methyl green; |
| narcëin—pyronin—methyl green; |
| narcëin—pyronin—methylene blue. |
2. Staining mixtures of 2 acids and 1 base, in particular the mixture to be described later in detail of
| orange g.—acid fuchsin—methyl green. |
| Further narcëin—acid fuchsin—methyl green, |