70 W. H. E M I G 



presents a general stain while the material taken from chrom-acetic 

 yields a nuclear stain. Similar results are obtained with iron 

 Haematoxylin. Should sublimate-acetic be used as the fixative, the 

 results differ : a general stain is obtained whenever the material is 

 left in the dye only long enough to provide the color pigment; by 

 overstaining in Haematoxylin and destaining in iron alum, the re- 

 sult is a nuclear stain. 



Staining with Haematoxylin. The procedure in which the re- 

 action is checked as soon as sufficient color has been produced is 

 known as the progressive method of staining. Alum Haematoxy- 

 lin is a progressive stain. The ripened solution stains animal 

 tissues within five minutes, plant tissues in 15 to 30 minutes. 

 Should the material be overstained in a few minutes, the solution 

 may be diluted with water. 



The regressive method of staining is followed when the dye 

 accumulates to excess, whereupon the surplus pigment must be re- 

 moved later with a suitable solvent or reducing agent. Iron 

 Haematoxylin is a regressive stain. For a rapid schedule, animal 

 tissues are mordanted in 1 per cent ferric alum 15 minutes, rinsed, 

 stained in ripened Haematoxylin one minute, differentiated in 0.25 

 per cent ferric alum for five minutes, rinsed, left in 0.01 per cent 

 ammonium hydroxide three minutes, and rinsed with distilled 

 water. With a longer schedule, the material is left in 2 to 4 per 

 cent ferric alum 12 to 24 hours, rinsed, stained 24 hours, differen- 

 tiated in 1 per cent ferric alum, rinsed, left in 0.01 per cent am- 

 monium hydroxide three minutes, and rinsed with distilled water. 

 Plant tissues are mordanted in 1 per cent ferric alum 30 minutes, 

 rinsed, stained in Haematoxylin three minutes, differentiated in 

 0.25 per cent ferric alum five to ten minutes, rinsed, left in 0.01 per 

 cent ammonium hydroxide three minutes, and rinsed with .distilled 

 water. A slower schedule requires 24 hours in the mordant and 24 

 hours in the stain; the blue-black pigment is so fast in root tips 

 there is no evidence of fading after twenty years. 



As a rule, a tissue prepared for cytological studies does not re- 

 quire a counterstain. Under low power objectives, a trace of color 

 in the cytoplasm aids in bringing out the third dimension of the 



