BINUCLEATE CELLS IX TISSUE CULTURES. 73 



copper acetate, placed between the source of illumination and the condenser, when 

 artificial light was used, was found to be an advantage (Kite 19136, p. 149). 



In studying the grosser changes, such as the variation in shape of the nucleus, the 

 4 mm. Leitz apochromatic objective was used; for the finer details the Leitz ^\y oil- 

 immersion objective was found to be satisfactory. Oculars were Leitz Compen- 

 sating Nos. 4 and 6. 



For observation of the living cells the cultures of the second day were generalh' 

 the most favorable; the growth was then usually abundant and the cells in a 

 healthy condition, with a fair proportion of mitoses. 



It is quite evident that continuous observation of the living cell, provided it 

 can be carried out successfully, is the ideal method of studying the sequence of 

 changes occurring therein. Indeed, for the study of amitosis it has been regarded 

 as indispensable, as witness the statement of Richards (1911, p. 125): "For ami- 

 tosis there is but one absolutely certain criterion, the observation of living material 

 and subsequent study of material fixed under observation;" he adds, "this is, of 

 course, impossible in most cases." 



The method has already' been used in the study of living multinucleate cells of 

 tissue cultures, Lambert (1912a) having attempted to settle the question of the 

 origin of giant cells growing from explanted tissue by its aid, and the character of 

 the results attained through its use was sufficiently encouraging to warrant its 

 application to the problem in hand, though not altogether satisfactory in view of 

 the obvious difficulties. It was hojied, too, that these difficulties would be mini- 

 mized by the use of glycosaline media, which produced films of tissue sufficiently 

 thin for study in the living condition. 



It was first planned to ascertain the full history of the binucleate cell by 

 selecting a cell with a single nucleus and observing it continuousl}' on the stage in 

 the warm box till either the nucleus divided and formed a double-nucleated cell 

 or the cytoplasm became merged with that of another mononucleate cell to form 

 a single cell containing two separate nuclei. Observations upon this binucleate 

 cell were then to be continued until the ultimate fate of the double nucleus was 

 disclosed. Drawings were to be made from time to time with the camera lucida. 



This ideal was found to be impossible of realization, on account of the technical 

 difficulties. Cultures under continuous observation, exposed, as they were, to 

 strong light, often showed evidences of degeneration; even daylight seemed to 

 cause this and the use of ray filters did not altogether eliminate it. Degeneration 

 was noticed at times even when the plan was followed of making short observations 

 and immediately turning the light off, the culture remaining continuously on the 

 stage. 



Living cells show a marked tendency to migrate; hence the cell under observa- 

 tion had to be closely watched to j)revent its escape from the field of vision. Other 

 cells often wandered over the cell under inspection, and so interfered with the work. 

 Added to these difficulties is the length of time involved in the process, which 

 necessitates many hours — even days — of continuous observation. Then, too, the 

 minuter cell changes are very difficult to follow, even for short ]ioriods, the only 



