42 Methods in Plant Histology 



different treatment, and all the preceding variations might fail 

 miserably with the pollen tubes of cycads. This stain is so impor- 

 tant 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 

 paraffin 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 /A thick. If the ribbons are only 5 //, 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 arche- 

 gonia 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. 



7. Wash in water, 5 minutes. 



The water should be changed several times. If the washing is 

 not thorough, the differentiation will not be sharp. 



