26. THE BEHAVIOR OF 

 ISOLATED EMBRYONIC CELLS 



PURPOSE: To determine the structure and the behavior of embryonic cells isolated from 

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

 and differentiation. 



MATERIALS: 



Biological: Urodele embryos from cleavage to neurula. Anuran eggs and embryos 

 can be used but are not as satisfactory. 



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

 Carbon and carmine particles, finely divided. 

 Nile blue sulphate: 1/750, 000 in Standard Solution. 

 Solutions: 



Standard Solution - both hypo- and hypertonic concentrations. 

 Standard Solution plus 1% KOH, freshly made up and adjusted to pH. 



9. - 11. 

 Standard Solution made up without the CaCl^ (Ca-free Standard). 

 Potassium oxalate (0.4%) and sodiunn citrate (0.4%), used to oppose 



the solidifying action of calcium. 

 KCN: M/40 to M/640 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 



1. Remove the bulk of the jelly from a blastula or gastrula stage, leaving the 

 fertilization (vitelline) membrane intact. Place the embryo in 1% KOH in 

 Standard Solution and observe continually under the low power magnification. 

 When the cellular mass has become disarranged, remove it (within the mem- 

 brane) to fresh Standard Solution (without KOH). After a few minutes change 

 again to fresh Standard 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 coverslip elevated by two hairs or glass slivers. 

 Study under both low and high magnification and note internal Brownian move- 

 ment, pseudopodial formation, and general activity. (Compare with accom- 

 panying figures from Holtfreter's paper. ) 



2. 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 directions under "1" 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. 



* The author acknowledges with pleasure the suggestions made by Dr. Holtfreter in organizing this exercise. See new section #28 

 on "Dissociation and Reaggregation of cells. " 



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