1*5*^ EXPERIMENTAL CHICK EMBRYOLOGY 



5- Sallne-A^ar plus yolk extract (omitting all albumen): 



a. Separate the yolk from the albumen in about 50 to 100 cc. of chick 

 Ringer's, without rupturing the vitelline membrane. Pull off the chalaza 

 and such albumen as you can grasp with the forceps. With glass rod roll 

 the yolk over to remove any adherent albumen. Pass the yolk through 

 several changes of Einger'a, In each removing more of the albumen. 



b. When fully denuded of its albumen, pour off all the Ringer's, puncture 

 the vitelline membrane with sharp forceps, grasp it, and allow the yolk 

 to flow out into a sterile beaker. 



c. Add about 10 cc. of the albumen- free yolk to 20 to UO cc. of saline-Agar 

 "A", shake vigorously to mix. 



(Note: The unbuffered, pure yolk has a pH range of k.^ to 6.0, hence this fac- 

 tor must be considered in relation to the results. It would be Instruc- 

 tive to compare this with buffered saline "B".) 



6. Saline-Agar plus pure albumen (omitting all yolk): 



a. I'llx 10 cc. of pure egg albumen with from 15 to Uo cc. of chick Ringer's. 

 Shake thoroughly, and centrifuge (as above). 



b. Mix 1 cc. of the above (supernatant) mixture with 1 cc. of saline-Agar 

 "A" to make up the substrate. 



(This may be made up with the buffered "B" or the non- buffered "A" saline 

 solutions. ) 



7. Saline-Agar (or blood plasma) plus embryonic extract : 



a. Place 5 embryos, ages from 5 to 8 days, in 20 cc. of Panne tt-Compt on 

 saline and thoroughly crush. 



b. Centrifuge the embryonic mash and draw off the clear supernatant fluid. 



c. Mix the embryonic extract with 



1. Buffered saline-Agar "B" at i+0°C. or 



2. Blood plasma. 



NOTE: It is impractical for the average graduate student to test all of the above 7 media. 

 It is therefore recommended that they be used in the sequence given (above) as far 

 as time and facilities will allow. 



Preparation of the explant : 



(1) Incubate fertile eggs for 20 to 2*+ hours at 58°C. 



(2) Open an incubated egg into a finger bowl containing 100 cc. of sterile chick 

 Ringer's solution. Simply break the egg open as though it were to be fried, 

 taking care not to break the yolk. 



(5) With forceps, grab the chalaza or the yolk, and cut (with sharp scissors) 

 through the vitelline membrane, making a circle around the blastoderm about 

 ^ inch away from its border. By cutting in one direction and rotating the 

 yolk mass (with forceps) in the opposite direction, the blastoderm can be 

 quickly encircled. 



(k) Grasp the margin of the blastoderm with the forceps, and roll it back and away 

 from the yolk. When the blastoderm is entirely freed from the yolk mass, and 

 can be clearly seen, work a blunt dissecting needle between the blastoderm and 

 the thin, transparent vitelline membrane. Now transfer the blastoderm, by 

 means of a wide-mouthed pipette, to a Petri dish containing about 20 cc. of 

 sterile chick Ringer's solution. 



(5) With fine dissecting (glass) needles trim away all of the yolky-opaque area. 

 The early embryo is now ready to be cultured, in whole or in part. 



Embryonic parts to be used as explants : 



Stages to be used: 



a. Definitive primitive streak 



b. Head-process stage 



c. Head-fold stage 



d. One to 6 somite stage 



