FREEHAND SECTIONS 89 



they should remain for at least 30 minutes— an hour, or even over- 

 night, does no damage. Pour off the alcohol and pour on a 50 per cent 

 alcoholic solution of safranin (a 1 per cent solution of safranin in 50 per 

 cent alcohol). Stain overnight, or even for 24 hours. 



Pour off the safranin (which may be used repeatedly) and pour on 

 50 per cent alcohol. The alcohol will gradually wash out the safranin, 

 but this stain is washed out more rapidly from cellulose walls than 

 from those which are lignified. The sections should remain in the al- 

 cohol until the stain is nearly — but not quite — washed out from the 

 cellulose walls, while still showing a brilliant red in the large lignified 

 tracheids. If 5 or 10 minutes in the alcohol draws the safranin from 

 the lignified walls as well as the cellulose, stain longer; if the differenti- 

 ation is not secured in 5 or 10 minutes, a small drop of hydrochloric 

 acid added to the alcohol will hasten the process. Some recommend 

 staining for only 1 or 2 hours, but the washing-out process is likely to 

 be rapid and uncertain. 



Pour off the alcohol and wash the sections thoroughly in ordinary 

 drinking-water. The washing should be particularly thorough if acid 

 has been used to hasten the previous process, for the preparations will 

 fade if any acid remains. 



Stain in Delafield's haematoxylin 3-30 minutes. Usually 5 minutes 

 will be about right. Delafield's haematoxylin will stain the cellulose 

 walls, but will have little or no effect upon lignified structures. 



Transfer to drinking-water, not distilled water. The red color of the 

 whole section, as it appears to the naked eye, will be rapidly replaced 

 by a rich purple. Continue to wash in water for 2 or 3 minutes after 

 the purple color appears. If the cellulose walls show only a faint pur- 

 plish color, put the sections back into the stain and try a longer period. 

 If the color is a deep purple or nearly black, add a little hydrochloric 

 acid (1 drop to 50 c.c. is enough) to the water. It is better to put the 

 drop into a bottle of water and shake thoroughly before letting the 

 acidified water act upon the sections. As soon as the sections begin to 

 appear reddish, which may be within 4 or 5 seconds, pour off the 

 acidified water and wash in drinking-water, changing the water 3 or 4 

 times a minute, until the reddish color caused by the acid has been re- 

 placed by the rich purple color so characteristic of haematoxylin. The 

 acid not only secures differentiation by dissolving out the stain from 

 lignified structures more rapidly than from cellulose walls but it also 

 removes the disfiguring precipitates which almost invariably accom- 



