58 METHODS IN PLANT HISTOLOGY 



The first American safranins were very unsatisfactory; but there 

 have been great improvements, and one company, the National AniUn 

 and Chemical Company, has produced a safranin with 90 per cent dye 

 content, much stronger than any of the European stains. This is the 

 safranin which has been certified by the Committee on Standardiza- 

 tion of Stains. Other companies are also improving. Coleman and 

 Bell's Safranin Y, for baciUi, is good for staining xylem. 



All safranins keep indefinitely, solutions 20 years old staining as 

 well, or better, than when fresh. 



An anilin safranin may be made according to the general formula. 



The transfer to the stain depends upon the formula. If the stain is 

 aqueous, transfer to the stain from water; if made up in 50 per cent 

 alcohol, transfer to the stain from 50 per cent alcohol. 



Sections of woody tissues, cut from living material, should be put 

 into 95 per cent alcohol for about 1 hour and then transferred to the 

 alcoholic stain. If cut from formalin material, sections should be left in 

 water for | hour, changing the water several times; then in 15, 35, and 

 50 per cent alcohol, about 5 minutes in each grade; and then be stained 

 in alcoholic safranin. If cut from formalin alcohol acetic-acid material, 

 the sections should be placed in 50 per cent alcohol for at least 10 

 minutes, changing once or twice before being placed in the alcoholic 

 stain. These are minimum times: longer times are just as good and 

 may be better. 



The time required for staining varies with the tissue, the fixing 

 agent, and the quality of the stain. In general, it may be said that 2 

 hours is a minimum and 24 hours a maximum. If the staining be too 

 prolonged, delicate structures, like starch grains, crystals, and various 

 cell constituents, may wash out. The mere fact that the whole section 

 does not wash off does not mean that everything is fastened to the 

 shde. On the other hand, with a short period, it is difficult to get a 

 sharp differentiation. In staining a vascular bundle, one should be 

 able to wash the safranin from the cellulose walls and still leave a 

 brilliant red in lignified structures. For paraffin sections, 3-6 hours 

 will usually be sufficient. It is a good practice to put the shdes into 

 the stain in the morning and finish the mounts any time in the after- 

 noon. For freehand sections of woody tissues, 24 hours is not too long, 

 and in a 50 per cent alcoholic safranin, the sections may be stained for 

 a week. 



From the stain, transfer to 50 per cent alcohol. If the sections are 



