208 BEHAVIOR OF ISOLATED CELLS 



If one can select a mesectoderm cell for particular atudy, the differentiation ia 

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

 .by temporary immersion in hypertonic solutions. (See figures from Hbltfreter'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 lyier (l9'*-6). The isolated cells are placed on the 

 center of a coverslip and inverted (in the hanging drop) over another coverelip 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 minutes, and then the coverslips are to- 

 gether 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 ]Cf^ alcohol in a Columbia staining 

 dish made for coverslips. From this point on the usual cytological procedures can be fol- 

 lowed, staining the smears with Feulgen for thymonucleic acid; Harris' haematoxylln for 

 gross chromosome structure; pyronln for ribosnucleic acid, etc. 



DISCUSSION : 



This exercise has been organized from a series of investigations by Hbltfreter (l9'*-5- 

 I9I4.7) which represent a new approach to the problems relating to morphogenetic movements. 

 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 un- 

 coated 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. 



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

 There are four major parts: 



1. Central core of seml-llquid endoplasm (plaamosol) which contains the nucleus, 

 yolk, lipo-proteln granules, melanin granules, and cytoplasmic ground sub- 

 stance. 



2. Caps\LLar wall of endoplasm, the plasmogel. 



5. Outer shell of fluid ectoplasm which contains smaller particles. This is 

 generally mlscible with water, and is rather thick. 



h. Thin refractive surface membrane, the plasmalemma, which forma irregular sur- 

 face bulges. This is semi-solid. Movements are initiated and executed by 

 forces localized in this layer or membrane but "they may be associated with 

 local solatlon and re-gelation of that portion of the endoplasm which under- 

 lies a fully developed ectoplasmic bulge." (Holtfreter) 



The adhesiveness of isolated embryonic cells is associated with the fluid environment, 

 the developmental stage of the cells, and the cytological type of cell involved. Cells 

 in isolation tend to lose their adhesiveness, and the hyaline bulges of the moving cells 

 are less adhesive. Adhesion is definitely toward other cells rather than toward the sub- 

 stratum such as glass. Cytolizlng cells become non-adhesive and are generally expelled 

 from an aggregation of cells. 



Any living cell which exhibits amoeboid movement, forming lobopodia, would be expected 

 to phagocytize particles from the environment. Some of these embryonic cells are more ef- 

 ficient than others, the difference being the more apparent in cells from the neurula 

 stage. 



Holtfreter has been able to keep isolated embryonic cells of the Amphibia alive and 

 active for weeks. There is around each cell an elastic surface coat whose strength in- 

 creases during development (differentiation) and whose existence is important in the be- 

 havior, survival, and differentiation of that cell. As long as this surface membrane is 

 Intact the cell is protected. 



