52 Methods in Plant Histology 



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 according to 

 the general anilin oil formula, transfer to the stain from water or, 

 if coming down from higher alcohols, from 35 per cent alcohol; if 

 the mixture of aqueous and alcoholic safranins is used, transfer from 

 35 per cent alcohol, if going up in the series, and from 70 per cent 

 alcohol, if coming down from stronger alcohols. For freehand 

 sections of woody tissues we always use the mixture. If sections are 

 cut from living material, leave them in 95 per cent alcohol for half 

 an hour and then transfer to the stain. Sections cut from alcoholic 



V 



material may be transferred directly to the stain. If cut from 

 formalin-alcohol material, leave the sections in 50 per cent or 70 

 per cent alcohol for ten minutes before transferring. If cut from 

 formalin material, leave them in water for 10 minutes, then in 35 

 per cent alcohol for 10 minutes before staining. 



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 slide. On the other hand, 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 to 6 hours 

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



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

 are deeply stained, and sufficient differentiation is not secured within 

 5 or 10 minutes, a drop of hydrochloric acid added to 50 c.c. of the 

 alcohol will hasten the extraction of the stain. If staining vascular 

 tissue, draw the stain from the cellulose walls, but stop before the 



