STAINS AND STAINING 47 



schedule for a root-tip and an embryo sac ; an alga might require differ- 

 ent 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 ac- 

 quainted with the general outline of the process and then adapt every 

 step to the case in hand. 



For the sake of illustration, I asked a prominent cytologist, Dr. S. 

 Yamanouchi, who has been notably successful in staining mitotic fig- 

 ures, to write a schedule indicating his methods of using this stain. 

 While he protested that the practice could not be written down, he 

 kindly prepared the following schedule, not for the instruction of his 

 colleagues, but to introduce the method to beginners. The schedule is 

 for paraffin sections. Throughout the 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 consider- 

 able 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 fx thick. If the 

 ribbons are only 5 /j. tliick, 200 slides can be treated. 



2. Xylol and absolute alcohol, equal parts, 5 minutes. 



3. Absolute alcohol, 5-7 minutes. 



4. Ninety-five, 85, 70, 50, and 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, 10-15 c.c. of hydrogen peroxide may be 

 added to 100 c.c. of the 50 per cent alcohol, where the sUdes should re- 

 main until the blackening disappears. 



5. Water, 10-20 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 cliromosomes 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. 



