FREEHAND SECTIONS 
91 
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. 
Safranin and Gentian-Violet.—This is a good combination for 
vascular anatomy. Stain over night in safranin, rinse in 50 per cent 
alcohol until the stain is reduced to a light-pink color in the cellu¬ 
lose walls; then rinse in water and stain 5 to 10 minutes in aqueous 
gentian-violet. Rinse in water, dehydrate in 95 and 100 per cent 
alcohol, 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 to 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. 
The bordered pits of conifers, the Bars of Sanio, and the middle 
lamella are beautifully stained by this method. 
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 
