BINUCLEATE CELLS IN TISSUE CULTURES. 77 



each 111 cells; thus the binucleate cells made up 0.9 per cent of the total cells 

 appearing in the new growth. 



Even in different preparations from the same tissue binucleate forms were 

 found with varying frequency. Among the 20 cultures of heart mentioned above, 

 one preparation showed 1 double nucleus to each 28 cells, while in another the ratio 

 was 1 to 1,180. 



Age of tissue, too, in these 20 heart preparations, had a bearing upon the inci- 

 dence of binucleate forms, new growths from the younger hearts showing a some- 

 what greater proportion of double nuclei than those from older cardiac tissue. In 

 hearts from chicks of 5 days' incubation there was, on the average, one binucleate 

 to each 105 cells; in 7-day hearts the ratio was 1 to 123, and in 8-day hearts it was 

 1 to 233. 



Finally, duration of growth seemed to be related to the relative frequency of 

 occurrence of these cells. In the same 20 j^rej^arations it was found that cultures 

 of the first 24 hours showed one double nucleus to each 183 cells; in cultures of the 

 second 24 hours the ratio was 1 to 86 cells. This seems to point to a considerable 

 amount of nuclear splitting in the second 24 hours, of which some at least probably 

 occurred within the new growth. Cultures of older duration were, in the slides 

 counted, not sufficiently numerous and tjqiical to base accurate conclusions upon. 



MORPHOLOGY. 



The average binucleate cell (figs, la, 7, and 9) is somewhat larger than the 

 average mononucleate, the area occupied by the nucleus being approximately twice 

 as great. Each nuclear part is, in size, shape, and general apiicarancc, very similar 

 to the nucleus of the mononucleate cell. The nuclear parts are often pressed close 

 together (figs, la, 60) and their adjacent surfaces are consequently flattened, the 

 intranuclear pressure in each being evidently equal. When thus related, the appear- 

 ance of the double nucleus in the living preparation (and indeed in some of the fixed 

 preparations) simulates a single nucleus which looks as though it were separated by 

 an equatorial membrane. Such an appearance has been interpreted as a nuclear 

 plate, or intranuclear membrane, and so described by Child (1904, 19075, and 1911, 

 p. 283), and others; but for reasons which will appear later on, I believe that such 

 appearances in tissue cultures are due to the apposition of nuclear surfaces, as above 

 described. 



In an elongated nucleus which has become bent upon itself the folded free 

 edge of nuclear membrane, projecting into the karyoplasm, may simulate a partition 

 which seems to be growing across the nucleus from one side to the other. A nuclear 

 configuration of this character is presented in Child's (1911) figure 16, page 293, and in 

 other of his figures. It is not to be wondered at that the approximated areas of 

 nuclear wall at the folded edge are somewhat attenuated and appear thin, as Child 

 (1911) has observed (p. 283). Such reduplications of nuclear membrane are not to 

 be looked upon as intranuclear membranes which cleave the nucleus ])y growing 

 across its equator. I have seen no evidence of a type of amitosis of this kind. 



Sometimes an equatorial membrane is simulated by an elongated nucleolus 

 lying across the nucleus. Again, as Richards (1911, p. 124) suggests: "A strand of 



