SPERMATOPHYTES— ANGIOSPERMS 351 



experiments with root-tips of Trillium sessile, obligingly furnished by 

 my friend, Dr. R. C. Spangler, who fixed the tips, shortly after noon, 

 in chromo-acetic-osmic solutions. With a stock solution of chromic 

 acid 1 g., and acetic acid, 1 c.c, osmic acid was added in various pro- 

 portions—! c.c. up to 10 c.c. to 100 c.c. of the stock solution. Fixing 

 was excellent in all; but staining was best with 6, 7, or 8 c.c. of osmic 

 to 100 c.c. of the solution. A long series of trials on onion root-tips 

 indicated that, with the acetic acid raised to 2 c.c, the results were 

 better. We recommend chromic acid 1 g., acetic acid 2 c.c, 1 per cent 

 osmic acid 6-8 c.c, and water 90 c.c For convenience, this may be 

 called the Chicago formula. Try the various fixing agents. Remember 

 that no matter how good the fixing may be, material can be rumed in 

 dehydrating, in clearing, or in the bath. Make yourself master of 

 Haidenhain's iron-alum haematoxylin ; then add the safranin, gentian 

 violet, orange combination; then safranin and anilin blue; and then 

 experiment for yourself, but remember that the triumphs of modern 

 cytology have been won with iron-haematoxyhn and that you cannot 

 read intelligently the literature of the past twenty-five years until you 

 have gained at least an approximate mastery of this stain. Of course, 

 dehydration, clearing, and infiltration must be very gradual. The 

 schedule by Yamanouchi on page 47 will repay careful study. 



When chromo-acetic-osmic solutions have been used for fixing, 

 there is a relation between the time needed in the second iron-alum 

 and the amount of 1 per cent osmic acid. If the time required for 

 differentiation in 2 per cent iron alum is less than 30 minutes, the per- 

 centage of osmic acid was too low; if more than 2 hours, it was too 

 high. About 1 hour in 2 per cent iron-alum, followed by 20-30 minutes 

 in 1 per cent (or weaker) is about right. It is somewhat like an expo- 

 sure in photography, except that an overexposure develops too fast, 

 while an overfixing in osmic acid comes out too slowly. If the iron- 

 alum haematoxylin is preceded by an overnight stain in safranin, and 

 the safranin is drawn until it has almost disappeared from the chromo- 

 somes, the figures will look as if stained only in iron-alum haematoxy- 

 lin, but the nucleoli will show the red and the cytoplasm will have a 

 slight tinge of pink. 



In staining with safranin, gentian violet, orange, allow the alcoholic 

 safranin to act for 16-24 hours; then extract it with 50 per cent alcohol, 

 slightly acidulated with hydrochloric acid, if necessary, until the stain 

 has almost disappeared from the spindle; then pass through 70, 85, 95, 



