BINUCLEATE CELLS IN TISSUE CULTURES. 75 



perature as the culture and of about the size of the hanging drop) was added to the 

 latter, the excess fluid being withdrawn. The dilution of the stain was thus approxi- 

 mately 1 in 200,000. The culture was immediatel}' examined under the microscope. 

 The dye rapidly diffused through the cytoplasm into the nucleus, the nucleoplasm 

 taking on a finely granular appearance; this latter was, apparently, the result of the 

 coagulative action of the dye. The nucleoli were distinctly marked out, and stained 

 much more darkly than the nucleoplasm or cytoplasm. The nuclear membrane, 

 too, was sharply outlined as a dark violet ring. This staining was very valuable in 

 delineating indistinct nuclear boundaries, since these, in the living unstained 

 culture, are often obscure. Irregularities in outline, such as indentations, were 

 rendered very plain, and the method was of assistance in studying the relationship 

 to one another of double nuclei. 



The cytoplasm, after this treatment, consisted of coarse violet granules in a 

 very faintly stained matrix, showing at times a slightly fibrous structure. Cell 

 borders were well marked, especially the pseudopodia, which, however, lost their 

 power of movement upon being stained. Intercellular bridges could be studied. 

 Mitochondria were not specifically stained, and degenerated in a short time. In a 

 culture so stained evidence of life, such as pseudopodial and mitochondrial move- 

 ment, cell migration, and mitosis, ceased almost at once, and in a few minutes 

 vacuoles formed in the cells and the entire culture became degenerate. 



Though gentian violet staining is of great assistance in obtaining a conception 

 of the morphology of the cells rapidly, under the conditions of the experiments its 

 toxic action precludes the possibility of the stained cell undergoing vital changes. 

 Owing to its coagulative action the appearance of the living protoplasm is not 

 accuratelj' reproduced in the stained preparation. In spite, however, of these 

 disadvantages, the use of gentian violet enables one to inspect portions of the cells 

 which, in the living condition, are almost, or quite, invisible, and also to examine 

 more accurately and easily some of the visible parts. 



M. R. and W. H. Lewis (1914 and 1915, p. 376) used janus green as a vital stain 

 in tissue cultures growing in Locke solution, and found that, although the mitochon- 

 dria were specifically stained, the dye was toxic in as low dilutions as 1 in 200,000, 

 and caused speedy death of the cells, as well as distortion of the mitochondria. 



Janus green (Hoechst), di-ethyl saffranin azo di-methj'lanilin, in Locke's solu- 

 tion, in a strength of 1 in 40,000, was applied to living cultures in the same manner 

 as the gentian violet, and was found to stain the mitochondria specifically in about 

 5 minutes, but no movements of these bodies could then be noted, and the threads 

 broke up into a row of granules. The cells soon died, as evidenced b}' their vacuo- 

 lated and degenerated appearance. 



While janus green staining provided a rapid and convenient method of observ- 

 ing mitochondria, its toxic action rendered it valueless as a means of studying vital 

 changes; moreover, the stained mitochondria soon lost their normal optical char- 

 acters, thus prohibiting extended observation. 



Some of these living cultures were fixed and stained. Osmic-acid vapor was 

 used for fixation, and Heidenhain's iron hematoxylin was found to give the best 

 staining. 



