50 METHODS IN PLANT HISTOLOGY 



week. Filter. Add 25 c.c. of glycerin and 25 c.c. of methyl alcohol. Al- 

 low to stand until the color is sufficiently dark. Filter, and keep in a 

 tightly stoppered bottle" (Stirling and Lee). The addition of the glyc- 

 erin and methyl alcohol will precipitate some of the ammonia alum in 

 the form of small crystals. The last filtering should take place 4 or 5 

 hours after the addition of the glycerin and methyl alcohol. 



The solution should stand for at least 2 months before it is ready for 

 using. This "ripening" is brought about by the oxidation of haema- 

 toxylin into haematin, a reaction which may be secured in a few min- 

 utes by a judicious application of peroxide of hydrogen. However, we 

 prefer to let the haematoxylin ripen naturally. There is no objection 

 to making this stain in considerable quantity, since it does not deterio- 

 rate. We have used Delafield's haematoxylin which had been in a cork- 

 stoppered bottle for 20 years, and it still gave the rich characteristic 

 stain. 



Transfer to the stain from 50 or 35 per cent alcohol or from water. 

 The length of time required is exceedingly variable. Sometimes sec- 

 tions will stain deeply in 3 minutes, but it is often necessary to stain 

 for 30 minutes or even longer. This stain may be diluted with several 

 times its own volume of water; when this is done, the time required is 

 correspondingly long, but the staining is frequently more precise. The 

 length of tune required will be fairly uniform for all material taken 

 from the same bottle. This fact indicates that the washing process, 

 which follows killing and fixing, is an important factor; if the washing 

 has been thorough, the material will stain readily; but if the washing 

 has been insufficient, the material may stain slowly or not at all. The 

 washing is particularly important when the fixing agent contains an 

 acid. Transfer from the stain to tap water. Distilled water is neither 

 necessary nor desirable. Some writers recommend washing for 24 

 hours, but this is entirely unnecessary; for paraffin sections on the 

 sHde, 5 or 10 minutes is long enough, and even for rather thick free- 

 hand sections 20 or 30 minutes is sufficient. Use plenty of water and 

 keep changing it as often as it becomes in the least discolored. Precipi- 

 tates are often formed when shdes are transferred directly to alcohol 

 from this stain, and sometimes even after washing in water. A few 

 gentle dips in acid alcohol (2 drops of HCl to 100 c.c. of 70 per cent 

 alcohol) will usually remove the precipitates. This extracts the stain 

 more rapidly from other parts than from the nuclei, and hence gives a 



