76 BINUCLEATE CELLS IN TISSUE CULTURES. 



The culture, growing in the hanging th-op, was removed froin the vaseUned 

 slide and exposed to the fumes from a 2 per cent aqueous sohition of the tetroxid 

 of osmium. Thi.s may be done b\' placing the cover-slip, drop down, over the mouth 

 of a bottle containing the fixative (the vaseline adhering to the cover-slip and pre- 

 venting the escai)e of the vapor) or, as suggested by ]\I. R. Lewis, by floating the 

 cover-slip, drop up, upon the osmic solution. Fixation is complete in 5 or 6 minutes, 

 and the preparation is then dark brown or black. It is now rinsed off with distilled 

 water, and pai^sed raj)idly tlirough ethyl alcohol solutions of 35, 50, and 70 per cent. 

 To the latter a few drops of hj'drogen jjeroxid are added, which bleaches the prepa- 

 ration. It is then passed back rapidly through the same alcohols, rinsed in distilled 

 water, and washed in running tap-water for 5 minutes. Too long immersion in 

 alcohol will cause the mitochondria to become dissolved out. 



The cover-slij), cultiu'e downward, is now floated upon a solution of 4 per cent 

 iron alum and allowed to remain for 12 to 24 hours; next it is washed in running tap- 

 water for 5 minutes and then immersed in 0.5 per cent aqueous hematoxylin for 

 24 to 48 hours, after which it is washed in running tap-water for 1 minute, differ- 

 entiated in 2 i)er cent iron alum and again washed in tap-water for 10 minutes, 

 dehydrated through the alcohol series, cleared in xylol, and mounted in balsam. 

 The hematoxyhn solution is prepared as follows: Hematoxjdin (10 per cent in 

 absolute alcohol), 0.5 c.c; distilled water, 10.0 c.c. 



These fixed i)rei)arations were used to make clear the morphology of the living 

 cells, especially such details as nuclear membranes, nucleoli, mitochondria, and cen- 

 trosomes. An attenijH was made to inck out the successive stages in the process 

 of direct division of the nucleus for comparison with the observations upon Uving 

 material, and thus to build up a series exhibiting the various changes. The phases 

 of mitosis were also studied, and drawings were made of interesting cells. For 

 statistical purposes cell counts were made of some of these preparations by jjlacing 

 a glass disk, upon which squares had been ruled, in the ocular, and using the 

 mechanical stage. 



THE BINUCLEATE CELL. 

 INCIDENCE. 



The frequency of occurrence of binucleate cells varies within wide limits in 

 cultures from different tissues. They were foimd to be most numerous in mem- 

 branes growing from the heart, and were not uncommon in cells of the connective- 

 tissue type from this and other tissues, but in the endodermal membranes from 

 stomach and intestine they were exceedingh^ rare. They may be even altogether 

 absent from the new growth. Lewis (1915, p. 156) notes that in growths from the 

 leg of chicks no amitotic forms were noted. 



To get an idea of the relative number of these cells as compared with the total 

 number of cells in the new growth, careful counts were made of 20 fixed cultures 

 from chick heart. Imperfect cells and those situated so close to the original piece 

 as to be indefinitelj' outlined were omitted. In these 20 preparations there was a 

 total of 41,725 cells, of which 375 were binucleate, or an average of 1 binucleate to 



