44 
METHODS IN PLANT HISTOLOGY 
the preceding chapter, but they are most effective when used after 
members of the chromic-acid series. 
Haidenhain’s Iron-Alum Haematoxylin. — This stain, introduced 
by Haidenhain in 1892 immediately gained great popularity and now, 
after more than 30 years’ constant use, still maintains first place in 
cytological investigations. Two solutions are used, and they are 
never mixed: 
A. 2 to 4 per cent aqueous solution of ammonia sulphate of iron. 
B. | per cent aqueous solution of haematoxylin. 
In making solution A, use the violet ferric crystals, hot the ferrous. 
The first solution acts as a mordant, i.e., it does not stain, but 
prepares the tissue for the action of the second solution. 
Solution A is at its best as soon as the crystals are completely 
dissolved and it remains in practically perfect condition for about 
two months, after which it gradually deteriorates. 
The haematoxylin crystals for solution B should be dissolved in 
water.. This will require about 10 days. The solution should then 
be allowed to “ripen” for 4 weeks before it is ready for use. Unfor¬ 
tunately, it remains at its best for only a short time, not more than 
5 or 6 weeks. This is because the “ripening,” which is an oxidation 
process, continues, and the solution becomes too ripe. During the 
ripening, a cotton plug should be used instead of a cork, to facilitate 
the oxidation; but as soon as the stain is ripe, a cork—preferably 
a perfectly fitting glass stopper—will prolong the maximum efficiency. 
Some prefer to dissolve the haematoxylin crystals in alcohol—-about 
10 g. in 100 c.c. of absolute alcohol. This solution should stand until 
it has a deep wine-red color. This will require 4 or 5 months, and a 
year is not too long. From this stock solution, make up small quanti¬ 
ties as needed. About 4 or 5 c.c. of this stock solution in 100 c.c. of 
water gives a practically aqueous solution, and it is already ripe. 
The American haematoxylin ripens faster and may reach its 
maximum efficiency in 2 or 3 weeks. One might naturally expect 
that it would retain its efficiency for a correspondingly shorter time; 
but we have found that, even for the structure of chromatin, the stain 
seems to stay at its best for at least two months. 
The general method is as follows: treat with A, stain in B, and 
then return to A to reduce and differentiate the stain. Never 
transfer directly from A to B, or from B to A; always wash in water 
before passing from one of the solutions to the other. 
