74 niNUCLEATE CELLS IN TLSSUE CULTURES. 



optical i)icture presented being very delicate shades of difference in refractivity. 

 The obscure and peculiar optical properties of living matter, as Kite (1913, p. 148) 

 points out, give rise to an important source of error. 



The ideal procedure having to be abandoned, the alternative practice was 

 adopted of following shorter periods of change and piecing tiu^ records of these 

 together. A start was made with the formation of the double nucleus, and here 

 another difficulty was encount(>red; it was manifestly impossible to tell which of 

 the thousands of nuclei in the culture was about to divide, and by selecting nuclei 

 at random, months might be spent without getting one which ultimately divided. 

 It was thus necessary to select a nucleus which gave some indication of being on 

 the way to di\ision, i. c, by elongation, or equatorial constriction; such a cell was 

 observed continuously until it divided or became degenerate. The subsequent 

 history was studied by selecting a double-nucleated cell and observing it con- 

 tinuously. 



In the observations the shape of the nucleus was particularly noted, and with 

 this was considered the behavior of the centrosphere, mitochondria, fat glolniles, 

 nucleoli, shape of the cell generally, and whether or not the cell itself ultimately 

 divided following nuclear division. Cells on or near the outer border of the new 

 growth were found most favorable, since they were larger and more free from sur- 

 rounding cells. Thej^ appeared to be usually quite healthy during the first 48 

 hours at least. 



As has here been noted, the morphology of the cell is difficult to make out in 

 the living and unstained condition, and it was thought that insjjection would be 

 much easier and more accurate if the details could be rendered visible by the use 

 of stains which would not impair the vitality of the cell. 



Churchman and Russell (1914) and Russell (1914) have recorded satisfactory 

 results with gentian violet in staining embryonic and adult tissues of the frog 

 growing in vitro. They stated that endothelium from adult frog pericardium in 

 frog's plasma to which gentian violet had been added grew definitely when the 

 strength of gentian violet was 1 in 2,000, and actively in a dilution of 1 in 20,000. 

 Furthermore, their records show that they were able to follow cell division in their 

 stained preparations. Clear karyokinctic figures were not seen in growing adult 

 frog tissue, but in embryonic frog tissue these figures were found in the dividing 

 stained nuclei. They beheve that the nucleus is stained intravitally and that 

 growth continues in the i^resence of the dye. Toxic action was shown when this 

 stain was used upon Paramecium, even in dilutions of 1 in 1,000,000. They believe 

 that "the use of stains in the plasma in which tissue is grown will ])robably facilitate 

 the study of nuclear growth." 



My results with gentian violet in chick tissue growing in Locke do not bear out 

 those of Churchman and Russell, for the staining could not be considered as in any 

 sense intra vitam, under the conditions existing in my experiments. 



A solution of Griibler's gentian violet was made uji in a strength of 1 in 100,000 

 with slighth' diluted saUne. Without removing the culture from the warm box, 

 the cover-slip was lifted off and a small drop of the stain (warmed to the same tem- 



