STAINS AND STAINING 
65 
Transfer from the orange G to 95 per cent alcohol, dipping the 
slide a few times in this merely to save the absolute alcohol. Dehy¬ 
drate in absolute alcohol 3 to 30 seconds. 
Clear in clove oil, as already described in the paragraph on gentian- 
violet. Transfer to xylol and mount in balsam. 
Safranin and gentian-violet are often used without the orange. 
In this case, transfer from the gentian-violet directly to 95 per cent 
alcohol, and proceed as before. 
An objection to both these methods is that the gradual series 
of alcohols cannot be used, because the gentian-violet washes out so 
rapidly. If one should try a filament of Spirogyra with either of 
these methods, it would hardly be recognizable when it reached the 
balsam; but with thin sections, especially when well fastened to the 
slide, conditions are different and there does not seem to be any 
serious damage. 
Just now, we are using a third method, which seems better than 
either of the two just described. 
C. Use the safranin solution described in B, an aqueous solution of 
gentian-violet, and a solution of orange G or gold orange in clove oil. 
Transfer to the safranin from 50 per cent alcohol and stain as 
directed under B. Rinse in 50 per cent alcohol for a minute, then 
in water for a minute, and stain in gentian-violet 3 to 30 minutes. 
Rinse quickly in water, dehydrate for about 5 seconds in 95 per cent 
alcohol and for about 10 seconds in absolute alcohol; then pour on 
the slide the orange dissolved in clove oil and allow it to act for 10 
to 30 seconds. Drain off the clove-oil orange, which can be used 
again, pour on pure clove oil and watch until the gentian is satisfac¬ 
tory, then transfer to xylol and balsam. 
With freehand sections, which are likely to be much thicker, 
the process is the same but the times will be longer. 
Safranin and Light Green.—Stain in aqueous safranin; wash in 
water; pass through 50, 95, and 100 per cent alcohol, about 2 minutes 
in each grade; stain in light green dissolved in clove oil; rinse in xylol 
and mount in balsam. 
If the stain does not rinse off readily, transfer to absolute alcohol, 
then to equal parts xylol and absolute alcohol, clear in xylol and 
mount in balsam. 
Cyanin and Ery thro sin.—Both solutions may be made according 
to the general formula for anilins, or 1 per cent aqueous solutions 
