46 METHODS IN PLANT HISTOLOGY 



pares the tissue for the action of the second solution. It is better to 

 make a 4 per cent solution and dilute it when a 2 per cent or weaker 

 solution is needed. The bottles and also the Stender dishes containing 

 the ammonia sulphate of iron nearly always become coated with a 

 yellowish film of iron oxide. This film also forms on sections or on 

 material to be mounted whole, but is so thin that it would be over- 

 looked unless one compared the sections with others without any film, 

 just as ordinary glass looks all right unless you compare it with fine 

 cut glass. This film forms only when the temperature of the fluid rises 

 above 18° C. So, keep the solution below 18° C. while using it. 



Solution A is at its best as soon as the crystals are completely dis- 

 solved 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 

 distilled water. This will require about 10 days, during which time the 

 bottle should be shaken often and vigorously. The solution must then 

 be allowed to ripen for a month before it is ready for use. During the 

 ripening, which is an oxidation process, 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 closefitting glass stopper — will prolong the 

 maximum efficiency. If kept in a cool place, away from strong light 

 and kept quiet, the stain may retain its efficiency for six months. 

 When needed, pour out very gently. The same solution, on a table in 

 the laboratory, poured out as one pours other stains, would lose its 

 efficiency in less than a month. As soon as the rich wine color begins 

 to disappear, the solution is worthless. 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. 



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 



