92 METHODS IN PLANT HISTOLOGY 



After sections have been cut, wash them in tap water, then in dis- 

 tilled water, and stain half an hour or more in weak Delafield's haema- 

 toxylin — about 5 parts of the solution, as given in the formula, to 100 

 parts of water — and then wash in distilled water. Stain in weak saf- 

 ranin — about 2 parts of the stock solution to 100 parts of water — 

 overnight or even for several days. Wash in tap water, then wash in 

 95 per cent alcohol for 30 seconds or longer, according to the appear- 

 ance of the stain. Dehydrate in absolute alcohol, clear in clove oil, 

 transfer to xylol, and mount in balsam. This method is very good for 

 gymnosperm woods. 



Since it usually happens that processes are commenced, but cannot 

 be completed, at a single laboratory period, it is necessary to know 

 where sections may be left for several hours or until the next day with- 

 out suffering injury. At 1, 2, or the pure water of 8 in the schedule 

 given above, sections may be left until the next day. If it is not de- 

 sirable to mount all of the sections which have been prepared, they 

 may be kept indefinitely in clove oil or xylol. If the sections are to re- 

 main for a year or more in the clearing agent, xylol is to be preferred. 

 Shells with good corks are best for keeping such material. 



For the study of vascular anatomy, this is the most permanent 

 stain which has come into general use. 



More recently, safranin combined with anilin blue or with light green 

 has been coming into favor. Both these methods will be described. 



Safranin and anilin blue. — Use the alcoholic safranin already de- 

 scribed, and a 1 per cent solution of anilin blue in 90 per cent alcohol. 



With this combination we should recommend a long stain in saf- 

 ranin, not less than 24 hours. Wash in 50 per cent alcohol, but do not 

 extract all the safranin from the cellulose walls. Stain 2-10 minutes in 

 anilin blue. Rinse a few seconds in 95 per cent alcohol, then treat for 

 about 5 seconds with 95 per cent alcohol slightly acidulated with hy- 

 drochloric acid— one drop in 50 c.c. of alcohol may be enough. The 

 weak blue should at once change to a bright blue and, at the same 

 time, the acid will remove some of the safranin. It is for this reason 

 that we proceed while the sections are still somewhat overstained in 

 safranin. Wash for 1 or 2 minutes in 95 per cent alcohol to remove the 

 acid. A trace of sodium carbonate, just enough to make the alcohol 

 alkahne, may be added to the 95 per cent alcohol. If any acid remains, 

 the safranin will fade. Dehydrate in absolute alcohol 5 minutes, clear 

 in xylol, or first in clove oil and then in xylol, and mount in balsam. 



