EXPERIMENTAL CHICK EMBRYOLOGY k29 



movements, and that the three major movements of convergence, invagination, and elongation 

 are found in both. The notochord and the floor of the neural tube arise from the material 

 of Hensen's node, as shown by deep vital dye staining. The anterior end of the primitive 

 streak (exclusive of Hansen's node) gives rise to the lateral walls and roof of the neural 

 tube, and to the somites. The anterior part of the head arises from prenodal materials. 

 Vital staining and charcoal marking are useful procedures in determining the prospective 

 significance of the various areas within the whole, normal, developing embryo. (Isolation 

 of these same areas onto culture media indicates that their prospective potencies are even 

 greater. ) 



THE METHOD OF VITAL STAINING: 



Chick embryo areas are not as simple to stain with vital dyes as are the comparable 

 areas of amphibian embryos. The fat globules and yolk often absorb some of the stain and 

 then disintegrate. Beyond a certain concentration, the living cells of the chick embryo 

 seem unable to tolerate the vital dyes. However, the staining must be fairly deep to be 

 significant for the primitive streak embryo is tridermic. In all instances the vitally 

 stained embryo must be removed to a watchglass, cleaned of all excess and adherent yolk, 

 and examined with transmitted light within 2h to hQ hours of the staining. 



Procedure : 



1. Prepare agar chips by soaking beaded agar in Nile Blue Sulphate (l/lO,000) and 

 also in Neutral Red (l/l0,000) and drying on glass plates. The agar beads will 

 take up the dye and, when dry, may be further cut down to any appropriate size 

 under a dissection microscope. Another method is to prepare some 2$ agar in 

 distilled water, add the dye (above concentration), pour the hot agar onto 

 glass plates and, when cool and dry, peel off the agar in thin and narrow strips 

 by means of a scalpel. These strips may then be cut to any sj,ze or shape. 



2. Wash instruments in warm soap and water, rinse, air dry and then place them In 

 80^ alcohol for sterilization. The instruments include watchmaker's (#5) for- 

 ceps, fine scissors, sharp scalpel, and hack saw blade. 



5. Locate the blastoderm of an 18-20 hour incubated egg, outline It on the shell 

 with pencil, and then place the egg (in the same position) in a finger bowl or 

 #2 Stender on abundant cotton. Orient the egg with the blunt end to the left. 



U, Following the procedure described above, make a window in the shell measuring 

 from 12 to 15 nm. in diameter. Do not injure the shell membrane. The window 

 should be within the circumscribed area, in the uppermost part of the shell. 



5. Place a drop of sterile Locke's (or saline) solution on the shell membrane, 

 and, when moist, rapture the membrane and remove it to the margins of the shell 

 opening. Avoid touching the underlying blastoderm and, if necessary, remove 

 any excess albumen with sterile pipette. 



6. With watchmaker's forceps place a small piece of blue (Nile Blue Sulphate) agar 

 (or even a particle of Nile Blue Sulphate powder) on the central region of the 

 blastoderm. The blastoderm, at this stage, is not readily visible but will be- 

 come apparent with blue staining. Eemove the blue agar after a few minutes, 

 i.e., after some of the dye has diffused into the embryo through the vitelline 

 membrane. (If powdered dye is used, this will be more difficult to remove.) 



Do not overs tain. 



7. Place a Neutral Red granule or red agar on the blastoderm in an anterior posi- 

 tion, i.e., anterior to Hensen's node, for a few minutes. Remove with forceps. 

 If this proves difficult, add a drop of sterile saline solution to float the 

 granule upwards, and then remove with watchmaker's forceps. 



8. Make a sketch of the blastoderm, any identifiable structures of the embryo, and 

 the positions of the colored areas. If a temporary window is placed over the 

 shell opening (coverslip) the sketch may be made during a brief candling. 



9. With melted, and soft paraffin seal a circular coverglass onto the egg surface, 

 over the window. This need not be made with the care of transplantations for 

 the duration of the experiment is short. 



