For practical purposes, besides the iodine and iodine-eosine solution described below (see page 46) the following are especially used:
1. =Hæmatoxylin solution with eosin or orange g.=
Eosin (cryst.) 0.5 Hæmatoxylin 2.0 Alcohol abs. Aqu. dest. Glycerine _aa_ 100.0 Glacial acetic acid 10.0 Alum in excess
The fluid must stand for some weeks. The preparations, fixed in absolute alcohol, or by short heating, stain in from half-an-hour to two hours. The hæmoglobin and eosinophil granules are red, the nuclei stain in the colour of hæmatoxylin. The solution must be very carefully washed off.
2. In the practical application of the triacid fluid, particular care must be taken, as M. Heidenhain first shewed, that the dyes are =chemically pure=[6]. Formerly granules, apparently basophil, were frequently observed in the white blood corpuscles, particularly in the region of the nucleus. They were not recognised, even by practised observers (_e.g._ Neusser) as artificial, but were regarded as preformed, and were described as perinuclear forms. Since the employment of pure dyes these appearances, whose meaning for a long time puzzled us, are but seldom seen.
Saturated watery solutions of the three dyes are first prepared, and cleared by standing for some considerable time. The following mixture is now made:
13-14 c.c. Orange-g. solution 6-7 c.c. Acid fuchsin solution 15 c.c. Aqu. dest. 15 c.c. Alcohol 12.5 c.c. Methyl green 10 c.c. Alcohol 10 c.c. Glycerine
These fluids are measured in the above-mentioned order, with the same measuring glass; and from the addition of methyl green onwards the fluid is thoroughly shaken. The solution can be used at once, and keeps indefinitely. The staining of the blood specimen in triacid requires only a little fixation, cp. page 35. The stain is completed in five minutes at most.
The nuclei are greenish, the red blood corpuscles orange, the acidophil granulation copper red, the neutrophil violet. The mast cells stand out by "negative staining" as peculiar bright, almost white cells, with nuclei of a pale green colour.
The triacid stain is very convenient. It is much to be recommended for good general preparations; =it is indispensable in all cases where the study of the neutrophil granulations is concerned=.