88 Methods in Plant Histology 



but is still brilliant in the lignified walls. If the stain acts for too 

 short a time, the washing-out process necessary to remove the stain 

 from the cellulose walls will leave only a pale-green color in the ligni- 

 fied walls. Stain in aqueous acid fuchsin for 2 to 10 minutes. This 

 should stain the cellulose walls sharply, but should not act long 

 enough to affect the lignified tissues. Pour off the stain (which may 

 be used repeatedly), and pour on 95 per cent alcohol, and imme- 

 diately pour it off and add absolute alcohol. The 95 per cent alcohol 

 should not act for more than 5 or 10 seconds, its only function being 

 to save the more expensive absolute alcohol. From 10 to 30 seconds 

 will usually be long enough for the absolute alcohol. Too long a 

 period in the alcohols will weaken the stain. Clear in xylol or clove 

 oil, and mount in balsam. 



If a 50 or 70 per cent alcoholic solution of iodine green has been 

 used, the stain should be washed out in 50 per cent alcohol; otherwise 

 the treatment is the same. 



Methyl Green (aqueous solution) and Acid Fuchsin is a good 

 combination, and the student may find it easier to get a good differ- 

 entiation than with iodine green. Follow the directions for the 

 aqueous iodine green and acid fuchsin. It may be necessary to 

 wash more rapidly, since the methyl green is easily extracted. 



Other Combinations might be suggested, e.g., iodine green or 

 methyl green with Bismarck brown, methyl green with Delafield's 

 haematoxylin; orange G .might be added after the safranin and 

 Delafield's haematoxylin, and various other stains might be tried. 

 In double staining it is usually best to combine a basic with an acid 

 stain. Green and red make a good contrast, but a section stained 

 with iodine green and safranin would be a failure, because both 

 stains would stain the xylem and neither would stain the cellulose. 

 Both stains are basic. Red lignin and green cellulose could be secured 

 by using safranin and acid green. Green ligniri and red cellulose, 

 as already indicated, can be got with iodine green and acid fuchsin. 



The Time Required for the different processes varies greatly, 

 and the time required for a subsequent process is often more or less 

 dependent upon the time given to processes which preceded it. 

 Good mounts of sections of the petiole of Nuphar advena have been 

 secured from material which had been cut, fixed, stained in safranin 



