ORIGIN OF AMPHIBIAN PIGMENT 267 



EECOED OF HETEROPLASTIC TRANSPLANTATIONS 



ISOLATION CULTURE OF THE NEURAL CREST 



Piecee of embryonic neural tube (Urodele stages #23 to #26) are stripped of their 

 dorsal ectoderm and then cut into small fragments, each with some neural crest, Jid are 

 cultured In isolation (Twitty & Bodensteln, 1959, Twitty, 19^5) to derive chromatophores. 

 There are two methods, and several culture media. 



The sterile culture media are: (1) Standard ( Holtfreter'e) Solution, in which the 

 bicarbonate is dissolved separately from the other salts and the two voliines are mixed 

 after boiling and cooling. (2) Coelomic Fluid which consists of fluid from the coelomic 

 cavity of the adult of the same species, removed under sterile conditions by means of a 

 sterile syringe. Twitty {19'+5) has found that females during the spawning season provide 

 the most abundant coelomic fluid. 



The culture methods are: (1) In a depression slide sealed over with a vaseline 

 ringed, coverslip. (2) On a coverslip inverted over a depression slide, the culture be- 

 ing in a hanging drop. (5) On a microscopic slide under coverslip slightly elevated by a 

 ring of soft paraffin. In each instance, the Isolated neural crest is in a llqiild en- 

 vironment, protected against evaporation and extrinsic change, and provided with facili- 

 ties for growth and expansion. The coelomic fluid is generally the best, for it provides 

 more than Just the isotonic salt requirements for maintenance and growth. Twitty and 

 Bodensteln (1959) found that boiling did not destroy the effectiveness of peritoneal fluid 

 in stimulating pigment development, but did reduce the risk of infection. 



The most successful Isolation cultures will probably be achieved if sterile coelomic 

 fluid is used and the neural crest Is Isolated Into this medium on a clean circular cover- 

 slip which is ringed with white vaseline. If the sterile Inverted depression slide is 

 brought down over the isolated tissue, pressed against the vaseline, and left in this 

 position for 2.k hours before re-inversion, the explant will become adherent to the cover- 

 slip so that when it (and the depression slide) is turned over, the neural crest and any 



