64 
METHODS IN PLANT HISTOLOGY 
B. The following formulas and method seem to be better for 
mitotic phenomena in plants: Make a 1 per cent solution of alcoholic 
safranin in absolute or 95 per cent alcohol, and after the safranin is 
completely dissolved, add an equal volume of a 1 per cent solution 
of aqueous safranin in water, thus making a 1 per cent solution of 
safranin in 50 per cent alcohol. Use a 1 per cent aqueous solution 
of gentian-violet, and a 1 per cent aqueous solution of orange G. 
Transfer paraffin sections to the stain from 95 per cent alcohol 
after the xylol or turpentine used in dissolving away the paraffin has 
been rinsed off. Stain 3 to 24 hours. If the period be too short, 
the washing out is so rapid that it is difficult to stop the differentiation 
at the proper point and, besides, the red is likely to be less brilliant. 
Rinse in 50 per cent alcohol until the stain is properly differentiated. 
Leave the slide in the 50 per cent alcohol until the stain is washed 
out from the spindle and cytoplasm, but stop the washing out before 
the chromosomes begin to lose their bright red color. If the washing 
out takes place too slowly, treat with slightly acidulated alcohol 
(one drop of HC1 to 50 c.c. of 50 per cent alcohol) for a few seconds. 
The acid must be removed by washing for 15 to 30 seconds in alcohol 
which has not been acidulated. 
Then dip the slide 5 or 6 times into water and stain in gentian- 
violet. The time required is so variable that definite instructions 
are impossible. The gentian-violet should stain the spindle, but 
not the chromosomes. If the stain be too prolonged, it may be 
impossible to get it out from the chromosomes and still leave it bright 
in the spindle. If the period be too short, the stain will wash out 
from the spindle. For mitotic figures in the germinating spores of the 
liverwort, Pellia, 30 minutes is not too long. In this case, the stain 
washes out easily from the chromosomes without the use of acid, 
and the spindle takes a rich violet which is not easily washed out. 
In embryo sacs of Lilium try 10 minutes. In pollen mother-cells try 
5 to 10 minutes. For root-tips try 2 to 10 minutes. Chromatin in 
the early prophases and in telophases will stain with the violet, and 
the violet will not wash out, but in phases in which fully formed 
chromosomes are visible the violet can be washed out if the period 
has not been too long. 
Remove the slide from the gentian-violet and dip it 5 or 6 times in 
water and then stain 30 seconds to 1 minute in orange G. The orange 
stains cytoplasm and at the same time washes out gentian-violet. 
