MORPHOGENETIC MOVEMENTS 423 



the prospective significance of the various areas within the whole, normal, developing 

 embryo. (Isolation of these same areas onto culture media indicates that their prospec- 

 tive 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 except that the dye is more visible. The fat globules and 

 yolk often absorb some of the stain and then disintegrate. Beyond a certain concentra- 

 tion, the living cells of the chick embryo seem unable to tolerate the vital dyes. How- 

 ever, 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 watch- 

 glass, cleaned of all excess and adherent yolk, and examined with transmitted light 

 within 24 to 48 hours of the staining. 



Procedure : 



1. Prepare agar chips by soaking beaded agar in Nile Blue Sulphate (1/10, 000) 

 and also in Neutral Red (1/10, 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 pre- 

 pare 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 size 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) forceps, fine scissors, sharp scalpel, and hack saw blade. 



3. 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. 



4. Following the procedure described above, make a window in the shell meas- 

 uring from 1 2 to 15 mm. 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, rupture 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 become apparent with blue staining. Remove 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 overstain. 



7. Place a Neutral Red granule of red agar on the blastoderm in an anterior 

 position, 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. 



* Gruebler's is best, but any dye used should be pre -tested because some samples have proven to be toxic to chick embryos. 

 Neutral red can be used but is less satisfactory. 



