268 



ORIGIN OF AMPHIBIAN PIGMENT 



The transplant should be treated as in the homoplastic experiments, the operation being 

 carried out in Urodele Operating Medium and after the graft has taken, transfer the host 

 to Urodele Growing Medium for further development. 



ISOLATION CULTURE OF THE NEURAL CREST 



Pieces of embryonic neural tube (Urodele stages #23 to #Zb) are^ stripped of their dorsal 

 ectoderm and then cut into small fragments, each with some neural crest, and are cul- 

 tured in isolation (Twitty & Bodenstein, 1939, Twitty, 1945) to derive chromatophores. 

 There are two methods, and several culture media. 



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

 bonate is dissolved separately from the other salts and the two volumes 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 (1945) 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 being in a 

 hanging drop. (3) On a microscopic slide under coverslip slightly elevated by a ring of 

 soft paraffin. In each instance, the isolated neural crest is in a liquid environment, pro- 

 tected against evaporation and extrinsic change, and provided with facilities 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 Bodenstein (1939) 

 found that boiling did not destroy the effectiveness of peritoneal fluid in stimulating pig- 

 ment 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 coverslip 

 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 

 24 hours before re-inversion, the explant will become adherent to the coverslip so that 

 when it (and the depression slide) is turned over, the neural crest and any derived cells 



Place in Petri dishes filled with Holtfreter's solution, 

 the tubes containing the explants are held in position 

 by partly imbedding them in a row ridge of vaseline. 

 The end near which the explant lies is left open; the 

 opposite end is plugged with vaseline to prevent any 

 flow of fluid through the tube which might be created 

 by tipping or disturbance of the dish. Semi-diagram- 

 matic drawing. 



A semi-diagrammatic drawing to represent 

 the differential onset of melaniration within 

 cultures of embryonic pigment cells devel- 

 oping in capillar/ tubes. 







From Twitty, 1944: Jour. Exp. Zool. 95:259. 



