BINUCLEATE CELLS IN TISSUE CULTURES. 



By Charles C. Macklin. 



INTRODUCTION. 



In examining a living tissue culture, or a preparation from the same, one fre- 

 quently finds a cell which contains two or more nuclei, of about equal size, slightly 

 separated or in contact. In a communication of Lewis and Lewis (1912 c, fig. 12) a 

 binucleate cell from a tissue culture is shown; more recently these authors (1915, 

 p. 391) have referred to the occurrence of such cells in tissue cultures under the 

 heading "Amitosis and giant cells." That they may be quite numerous in an area 

 of new growth is seen by referring to figure 1, where one quadrinucleate and six 

 binucleate cells appear in a small field. 



The question of their origin and fate in cultures of embryonic tissue, involving 

 as it does the idea of direct nuclear division, gathers interest from the fact that such 

 cells are found in embryonic tissue developing in vivo, and from the further fact that 

 they probably represent the first stage in the formation of certain giant cells. The 

 problem of inquiring into their history by prolonged observation of the living cell 

 was suggested by M. R. and W. H. Lewis. 



METHOD. 



Cultures were grown in ordinary hanging-drop i^reparations, the technique of 

 W. H. and M. R. Lewis (1911, 1912rt, 19126, 1915) being employed. The tissue 

 was obtained from embryo chicks of from three to ten days' incubation. Heart 

 tissue was most frequently used, and gave very satisfactory results. 



Locke solution as a culture medium was used. A stock saline solution was 

 first made up as follows: NaCl, 18 grams, 0.9 per cent; KCl, 0.84 gram, 0.042 per 

 cent; CaCU, 0.5 gram, 0.025 per cent; NaHCOs, 0.4 gram, 0.02 per cent; H2O, 

 2,000 c.c. Freshly distilled water and absolutely clean bottles are indispensable. 

 The solution will keep apparently good for months. 



Culture media was made up, from time to time, as required, from this stock 

 solution, in 100 c.c. lots, by dissolving from 0.25 to 1 gram of dextrose in 100 c.c. 

 of saUne, thus making a solution of 0.25 to 1 per cent of dextrose. The media was 

 then placed m clean plugged test-tubes, 10 c.c. in each, and steriUzed in the Arnold 

 sterilizer for 30 minutes, after which it was stored for use as required. It should be 

 made up fresh every two weeks. 



The best results were obtained by diluting this media, when it was being used, 

 by the addition of 20 to 25 per cent of freshly distilled, sterile water, since some 

 evaporation went on during the planting, and further concentration of the media 

 often occurred in the preparation from evaporation of the hanging drop and condon- 



