FIXIXa AM) STAIMXa M/CROSCOr/C OBJECTS 245 



they stay about 10 minutes, and anther fragments are then 

 removed. A long stay is injurious. These slides are then 

 run through 15, 30, and 50 per cent alcohols (about 5 

 minutes in each), up to 70 per cent. Here they are left 

 over night. They are then put for 24 hours in a 1 per cent 

 solution of ferric ammonia alum in 70 per cent alcohol. 

 Follows a brief washing in 70 per cent alcohol, and a soaking 

 of 15 minutes to 1 hour in 70 per cent alcohol. The 

 shorter the time, the more deeply the metaphase and cyto- 

 plasm stain. Then comes 2 to 24 hours staining in a solu- 

 tion of 0.5 gram of brazilin in 100 cubic centimeters of 70 

 per cent alcohol.^ Then the smears are washed briefly in 70 

 per cent alcohol, and differentiated in a 1 per cent solution 

 of iron alum in 70 per cent alcohol, for from 1 minute to 

 1 hour or more. The pachytene stage and the chromo- 

 meres usually require only slight differentiation with iron 

 alum, as 2 or 3 hours of staining usually suffices for them. 

 But when metaphases haye been stained for a longer time, 

 a differentiation of one or more hours is sometimes 

 required, especially with small chromosomes such as those of 

 Datura, Matthiola, or Nicotiana. 



After differentiation is seen to be satisfactory, by the 

 microscope, upon examining the preparations under cover- 

 glasses, the slides are washed in 70 per cent alcohol, and 

 transferred to 95 per cent alcohol, where they may be left 

 without injury for some hours. They are then passed 

 through (a) absolute alcohol, (&) a mixture of equal volumes 

 of absolute alcohol and thin cedar oil, and (c) a mixture of 

 equal volumes of xylol and thin cedar oil; and finally 

 washed with xylol. They are mounted by draining off 

 the xylol, applying a drop or two of immersion cedar oil, 

 and covering with a measured cover-glass. Such prepara- 

 tions show rapid fixation, except where sap from the anther 

 wall or connective has reached the pollen mother cells. 

 The brazilin stain, at its best, shows chromomeres, or 

 metaphase chromosomes, brown to black, and cytoplasm 



1 When using freshly made solution of brazilin, it is well to add one or 

 two drops of 1 per cent solution of iron alum to each 50 cubic ccsntimcters. 



