STAINS AND STAINING 
45 
While all follow the general method just indicated, no two investi¬ 
gators would prepare exactly the same schedule, even for staining the 
same object, e.g., root-tips; neither investigator would use the same 
schedule for a root-tip and an embryo sac; an alga might require 
different treatment, and all the preceding variations might fail 
miserably with the pollen tubes of cycads. This stain is so important 
that every worker must learn it, and the only way to learn it is to 
become acquainted with the general outline of the process and then 
adapt every step to the case in hand. 
For the sake of illustration, I asked two prominent cytologists, 
Dr. S. Yamanouchi and Dr. L. W. Sharp, both of whom have been 
notably successful in staining mitotic figures, to write schedules 
indicating their methods of using this stain. While both protested 
that the practice could not be written down, they kindly prepared 
the following schedules, not for the instruction of their colleagues, but 
to introduce the method to beginners. Both schedules are for par¬ 
affin sections. Throughout the first schedule, I have interpolated 
comments and suggestions. 
Yamanouchi’s Schedule.— 
1. Xylol, 5 minutes, to dissolve the paraffin. 
Do not heat the slides to melt the paraffin. However, a gentle 
warming which does not approach the melting-point of the paraffin does 
no damage and makes the paraffin dissolve more readily. The xylol 
soon has considerable paraffin in solution, but 100 c.c. of xylol should 
remove the paraffin from at least 100 slides with ribbons 25 mm.long and 
10 ix thick. If the ribbons are only 5 \x thick, 200 slides can be treated. 
2. Xylol and absolute alcohol, equal parts, 5 minutes. 
3. Absolute alcohol, 5 to 7 minutes. 
4. 95, 85, 70, 50, 35 per cent alcohol, 5 minutes each. 
If material has been fixed in a reagent containing osmic acid, it 
should be bleached. For this purpose, 10 to 15 c.c. of hydrogen peroxide 
may be added to 100 c.c. of the 50 per cent alcohol. 
5. Water, 10 to 20 minutes. 
If any alcohol is left in the sections, the staining will not be brilliant. 
Change the water several times. 
6. Iron-alum. 
Use the 4 per cent solution. For many objects, like the archegonia 
of gymnosperms and the embryo sacs of angiosperms, 1 hour is usually 
enough. For chromosomes in root-tips and anthers, 2 hours may be 
long enough; but for algae, 2 hours is generally a minimum. 
