216 BEHAVIOR OF ISOLATED CEUS 



The cells of a neurula should be isolated with 0. 1% KOH in Standard Solution and then 

 transferred to fresh Standard Solution (without KOH) to which some carbon or car- 

 mine particles have been added. Occasionally one will see the amoeboid-type of in- 

 gestion of the foreign particles, a process similar to normal phagocytosis. 



DIFFERENTIATION 



The neurula-stage isolated embryonic cells in Standard Solution may be placed in a 

 culture dish or a depression slide and sealed with a rim of vaseline around the cover 

 or coverslip. All conditions must be aseptic. The cells will often survive from 2 

 to 7 days and many will differentiate. 



If one can select a mesectoderm cell for particular study, the differentiation is most 

 graphic. These mesectoderm cells can be isolated from the closed neural tube stage 

 by temporary immersion in hypertonic solutions. (See figures from Holtfreter's 

 paper. ) 



The chemical isolation of neurula-stage cells may require several hours of exposure. 

 The isolation of blastula stage cells takes from 3 to 5 minutes. 



A rapid cytological examination of normal but isolated blastula, gastrula, or neurula 

 cells and of isolated cells of the neurula which have differentiated, can be achieved 

 by the technical procedure recommended by Tyler (1946). The isolated cells are 

 placed on the center of a coverslip and inverted (in the hanging drop) over another 

 coverslip on which there is a drop of Bouin's fixative. If the edges of the upper 

 coverslip are placed across the corners of the lower coverslip, they can later be 

 separated the more easily. The Bouin's fixative should be allowed to act for 5 min- 

 utes, and then the coverslips are together immersed in a Syracuse dish of Bouin's 

 fixative and the upper coverslip is gently separated from the lower one by means of 

 a needle. Allow the Bouin's fixative to act on the cell smears for another 5 minutes, 

 then transfer to 70% alcohol in a Columbia staining dish made for coverslips. Froni 

 this point on the usual cytological procedures can be followed, staining the smears 

 with Feulgen for thymonucleic acid; Harris' haematoxylin for gross chromosome 

 structure; pyronin for ribosnucleic acid, etc. 



DISCUSSION: 



This exercise has been organized from a series of investigations by Holtfreter (1943- 

 1947) which represent a new approach to the problems relating to morphogenetic move- 

 ments. Holtfreter has shown that up to a certain stage, any isolated cell of the embryo 

 is ready to unite with any other similar cell, provided the cells face each other with their 

 uncoated surfaces. Such isolated cells show an inherent tendency to movement due to the 

 autonomous activity of the cell membrane and not to any activity of the endoplasmic core. 

 There are wave-like contractions of the plasmalemma and an internal shifting of a clear 

 fluid mass which often results in the formation of lobopodia. Aggregation of cells re- 

 sults in the reduction of exposed surface tension. (See Section 28 on page 224). 



The general cytology of the amphibian cell is remarkably like that of the Amoeba. There 

 are four major parts: 



1. Central core of semi-liquid endoplasm (plasmosol) which contains the nucleus, 

 yolk, lipo-protein granules, melanin granules, and cytoplasmic ground substance. 



2. Capsular wall of endoplasm, the plasmogel. 



3. Outer shell of fluid ectoplasm which contains smaller particles. This is gener- 

 ally miscible with water, and is rather thick. 



