ORIGIN OF AMPHIBIAN PIGMENT 269 



will be merely the thickness of the coverslip away from microscopic examination. Such 

 cultures should last for 8-10 days, or longer at lower temperatures. 



Recently (1944) Twitty cultured pigment cells from neural crests within capillary tubes 

 measuring 0. 25 to 0. 45 in diameter. The neural crests were drawn into the tubes by 

 oral suction, while submerged in sterile Standard (Holtfreter ' s) Solution, and the tubes 

 were tilted slightly (within the Standard Solution) and the end of each tube farthest away 

 from the end containing the explant was embedded in non-toxic vaseline. This vaseline 

 acted as a plug, and at the same time held the tube in place. The tilting was achieved by 

 a mid-way ridge of vaseline, but this could be accomplished equally well with a knotched 

 paraffin ridge. The free end, i. e. , where the explant is located, was open to the culture 

 medium. During examination, the curved sides of the capillary tube acts somewhat as a 

 lens, magnifying the contained neural crest and pigment cells. Twitty found that pigment 

 formation in the capillary tubes was dependent upon substances diffusing from the nervous 

 tissue of the explant, and that the first cells to become pigmented were those found deep- 

 est within the tube. He attributed this to regional differences in the concentration of oxy- 

 gen and pigment precursors or oxidases essential to melanin formation. 



The record for this experinnent will consist of a series of daily drawings or photographs 

 of the explants and the cells (pigmented and otherwise) which are derived therefrom 



SELECTIVE STAINING OF IN-VIVO NEURAL CREST DERIVATIVES 



In transplantation and isolation experiments there is always the question as to whether 

 the derivatives of the excised anlage' are the same as they would have been in the original 

 site of the donor. Stone (1932) refined a method of preserving the vital dye Nile Blue 

 Sulphate in the fixed embrye so that it could be located in sectioned material. It is there- 

 fore in the nature of a confirmatory experiment that the following procedure is given. 



1. Select Urodele embryos of stage #23, and remove all of their membranes in Growing 

 Medium. Place them in an operating dish with a Permoplast or soft paraffin base, 

 and mould depressions to hold them with the neural folds uppermost. 



2. Cut a piece of Nile Blue dyed agar (0. 1 gr. Nile Blue Sulphate in 2% agar in 100 cc. 

 of distilled water, dissolved by heating, and poured, while hot, onto glass plates 

 covered with a very thin layer of glycerine. When dried, the agar can be peeled off 

 of the plate in thin sheets of any size or shape (e. g. , the shape and size of the neural 

 fold). Place this minute piece of agar flat on a piece of coverslip and pass the cover- 

 slip through a flame. This will cause the agar to melt slightly, and become adherent 

 to the coverslip chip. With practice one can provide a marker of the exact shape and 

 size of the neural fold. 



3. After the neurula stage (#23) is firmly placed within the depression, in Standard Solu- 

 tion, bring the (inverted) coverslip chip into position so that the Nile Blue Agar will 

 make precise contact with one of the neural folds. Press it against the embryo, and 

 anchor the edges of the glass chip in the surrounding Permoplast. Hold the neurula 

 in this position for 20-30 minutes, while the dye is being transferred to the neural 

 fold, and then gently remove it without tearing away any of the cells of the embryo. 



4. Transfer the embryo to Urodele Growing Medium for development until stage #28 or 

 later. The dye will remain for a long time and spread with the cells it has invaded, 

 so that it may be found even after the pigment has begun to appear. 



5. (a) Fix the embryo in Zenker-acetic for two hours; wash in running tap water 1 hour. 



(b) Place in 1% aqueous solution of phosphomolybdic acid for 2 hours (Lehmann, 1929). 



(c) Transfer for half hour periods through the ascending alcohols to each of which has 

 been added 0. 1% phosphomolybdic acid. This acid preserves the dye. 



