BEHAVIOR OF ISOLATED EMBRYONIC CELLS* 



PUBPOSE : To determine the structure and the hehavior of embryonic cells isolated from 



each other, with particular emphasis on their motility, adhesiveness, phagocytosis, and 

 differentiation. 



MATEBIAI5 : 



Biological : 



Urodele emhryos from cleavage to neurula. 

 used hut are not as satisfactory. 



Anuran eggs and embryos can be 



Technical : Operating needles, slides, coversllps, depression slides. 

 Carbon and carmine particles, finely divided. 

 Nile blue sulphate: l/750,000 in Standard Solution. 

 Solutions : 



Standard Solution - both hypo- and hypertonic concentrations. 

 Standard Solution pl'ia 1^ KOH, freshly made up and adjusted to pH. 



9.0 - 11.0. 

 Standard Solution made up vrithout the CaClg (Ca-free Standard). 

 Potassium oxalate (O.^i-^) and sodium citrate (0.1^^), used to oppose the 



solidifying action of calcium. 

 KCN: m/1+O to m/614-0 made up in Standard Solution. 



METHOD: 



Precautions : Use reasonably aseptic conditions, particularly in the differentiation 

 observations. Sterilization is not generally necessary. 



Controls : None are possible in this type of qualitative experiment. 



Procedure: 



MOTILITY 



Remove the bulk of the Jelly from a blastula or gastrula stage, leaving the fer- 

 tilization (vitelline) membrane Intact. Place the embryo In If) KOH in Standard 

 Solution and observe continually under the low power magnification. When the 

 cellular mass has become disarranged, remove it (within the membrane) to fresh 

 Standard Solution (without KOH). After a few minutes change again to fresh Stand- 

 ard Solution. Now rupture the fertilization membrane with sharp watchmaker's 

 forceps. This will liberate the cells which may then be picked up with a fine 

 pipette and transferred to a microscopic slide for examination beneath a cover- 

 slip elevated by two hairs or glass slivers. Study under both low and high mag- 

 nification and note internal Brownian movement, pseudopodial formation, emd 

 general activity. (Compare with accompanying figures from Holtfreter's paper.) 



Stain an entire embryo at any early stage, using Nile blue sulphate. Allow the 

 embryo to remain in the vital dye until its surface is distinctly blue in color. 

 Now follow liirections under "a" above. The peripherally exposed parts of cells 

 will be stained the more heavily and motility can be studied in relation to the 

 original polarity or axis of the cell. 



c. Repeat either 



or "b" but place the disarranged cells in calcium-free Stand- 



ard Solution and note the effect on amoeboid movement as well as cellular aggre- 

 gation. 



d. Cells may be separated from each other mechanically, with fine glass needles. 

 This should be attempted, particularly with the later (neurula) stages where the 

 germ layers can be distinguished. 



* The author acknowledges with pleasure the suggestions made by Dr. Hbltfreter in organiz- 

 ing this exercise. 



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