56 
METHODS IN PLANT HISTOLOGY 
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 
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, with a short period, 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 afternoon. 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 
lignified walls begin to fade. If a contrast stain is to be added, 
like light green, which weakens the safranin; or aniline blue or 
Delafield’s haematoxylin, which need to be followed by an acid; 
the safanin should be strong enough to allow the necessary reduction. 
If staining mitotic figures, draw the stain from the spindle, but stop 
before the chromosomes begin to weaken. When the desired differ¬ 
entiation has been reached, wash out the acid in 50 per cent alcohol, 
if acid has been used. About 5 minutes should be sufficient. 
