4^ EXPLANTING AND CULTIVATING 



up to a 10-20 somite stage provided one also explants the blastoderm endo- 

 derm layer up , upper surface against the medium. (See Biol. Bull I960, 

 119:338). Explants should be removed to dish of saline for examination 

 against a black background. 



5. Saline-Agar 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 al- 

 bumen 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 

 Ringer's, 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 40 cc. of saline-Agar "A", 

 shake vigorously to mix. 



(Note: The unbuffered, pure yolk has a pH range of 4. 5 to 6. 0, hence this factor 

 must be considered in relation to the results. It would be instructive to 

 compare this with buffered saline "B". ) 



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



a. Mix 10 cc. of pure egg albumen with from 1 5 to 40 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 3 embryos, ages from 5 to 8 days, in 20 cc. of Pannett-Compton 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 40°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 24 hours at 38°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. 



3. With forceps, grab the chalaza or the yolk, and cut (with sharp scissors) through 

 the vitelline membrane, making a circle around the blastoderm about 1/4 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. 



4. 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. 



