1. Haematoxylin (C16H14O6) is an ether extract of the wood of Hæmatoxylon campechianum, a tree found in the West Indies and Central America. In itself not a dye, it becomes one of the most valuable when oxidized to hæmatein (C16H12O6), and combined with alum, iron or other mordants to form a lake. It then stains nuclei a deep violet-blue or black color that is practically permanent. Mucin, lime-salts, bacteria, and colonies of actinomyces are also stained varying shades of blue. If the staining process is prolonged the entire tissue, as well as celloidin, becomes more or less heavily stained blue. A pure nuclear stain is obtained by interrupting the stain at the right time (examine in water); or if the sections are over-stained they may be differentiated in acid alcohol (1 per cent hydrochloric acid in 70 per cent alcohol). Hæmatoxylin stains well after all fixing-solutions except osmic acid; some of its staining-formulæ stain slowly after fixation in Zenker’s fluid. On the whole, it is by far the best general nuclear stain for laboratory and diagnostic work. It is employed in numerous staining formulæ, the most useful of which are given here. These formulæ differ chiefly in the time of staining, “ripening” of the stain (oxidation of hæmatein), intensity of stain, necessity of differentiation, etc.
a. Böhmer’s Alum-haematoxylin.
Dissolve 5 grms. of hæmatoxylin crystals in 50 cc. of absolute alcohol; add this drop by drop, while stirring, to 1,000 cc. of a 1 per cent solution of potassium alum. Expose in open vessel to air and light for 1-2 weeks. Filter before using.
b. Hansen’s Haematoxylin.
To 200 cc. of alum-hæmatoxylin solution brought to the boiling-point add 2 cc. of a concentrated solution of potassium permanganate. Cool quickly; filter when cold. Can be used at once without further ripening. It tends to stain diffusely.
c. Delafield’s Haematoxylin.
To 400 cc. of a saturated solution of ammonia alum add a solution of 4 grms. of hæmatoxylin in 25 cc. of absolute alcohol. Expose mixture to air and light for 3-4 days; filter; then add 100 cc. of glycerin and 100 cc. of 95 per cent alcohol, and filter. Expose to light until solution is dark enough, then keep in tightly-stoppered bottle. It is a strong stain, and may be diluted with distilled water when desired. The solution keeps well.
d. Ehrlich’s Acid-haematoxylin.
Dissolve 2 grms. of hæmatoxylin in 100 cc. of absolute alcohol. Add this to a saturated solution of potassium alum in water 100 cc., glycerin 100 cc., and glacial acetic acid 10 cc. Allow mixture to stand for a week exposed to air and light; then filter. Keep in well-stoppered brown bottles. The solution stains best when it is six months old, and may be kept for several years. It does not overstain, and on the whole is more useful than Böhmer’s, Hansen’s or Delafield’s.
e. Mayer’s Haemalum.