Stains and Staining 41 



the preceding paper, but they are most effective when used after 

 members of the chromic-acid series. 



Haidenhain's Iron-Alum Haematoxylin.- -This stain was intro- 

 duced by Haidenhain in 1892 and has gained a well-deserved popu- 

 larity with those engaged in cytological work. 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, not 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. 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 quantities 

 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 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. It is a good 

 plan to use a 4 per cent solution of A to precede the stain and a 2 

 per cent solution for differentiating. 



While all follow the general method just indicated, no two investi- 

 gators would prepare exactly the same schedule, even for staining the 

 same object, e.g., root-tips; neither investigator would use the same 

 schedule for a root-tip and an embryo-sac; an alga might require 



