FREEHAND SECTIONS 95 



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 com- 

 bination, and the student may find it easier to get a good differentia- 

 tion 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. 



Safranin and gentian violet. — This is a good combination for vascu- 

 lar anatomy. Stain overnight in safranin, rinse in 50 per cent alcohol 

 until the stain is reduced to a light pink color in the cellulose walls; 

 then rinse in water and stain 5-10 minutes in aqueous gentian violet 

 or crystal violet. Rinse in water, dehydrate in 95 and 100 per cent 

 alcohol, differentiate and clear in clove oil, transfer to xylol, and mount 

 in balsam. 



Orange may be added to this combination, making a triple stain. 

 In this case, do not reduce the safranin at all, but rinse quickly in 50 

 per cent alcohol, then in water, stain in gentian violet, rinse in 95 per 

 cent alcohol, and stain for 1 or 2 minutes in orange dissolved in clove 

 oil. This will not only reduce and differentiate the gentian violet, but 

 will reduce the safranin. Transfer to xylol and mount in balsam. If 

 the safranin is drawn out too rapidly, stain for 15-30 seconds in the 

 orange, transfer to clove oil without any orange until the gentian 

 violet is satisfactory; then transfer to xylol and mount in balsam. 



Both the violet and the orange may be used in clove oil. In this 

 case, transfer to the violet from absolute alcohol, and from the violet 

 to the orange ; then to pure clove oil until the violet looks right. Trans- 

 fer to xylol and mount in balsam. 



The bordered pits of conifers, the Bars of Sanio, and the middle 

 lamella are beautifully stained by this method. For the Bars of Sanio, 

 it may be necessary to stain in the clove oil violet over night, or even 

 for 24 hours. 



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 Dela- 

 field's haematoxylin, and various other stains might be tried. In dou- 

 ble staining it is usually best to combine a basic with an acid stain. 



