220 CULTURING ISOLATED ANIAGEN 



(Note: If one treats the early post-neurula embryo as a mosaic of many 



organ anlagen, explantation of a variety of structures may be studied. 

 It should be recorded, however, exactly what germ layers are in- 

 cluded in each explant. ) 



The embryo is first passed through several changes of sterile culture medi- 

 um to remove adherent bacteria, and then the anlagen are removed by means 

 of iridectomy scissors, lancets, glass needles, etc. and are transferred to 

 the culture medium by means of an adequately wide-mouthed, sterile pipette. 



4. Culturing of the explants : 



The explant may be placed directly into the appropriate medium in the watch- 

 glass surrounded by a moat of distilled water which provides a moist cham- 

 ber when the Petri dish cover is replaced. 



An alternative method* is to prescribe a small ring with a sterile glass rod 

 dipped into soft paraffin, ** the ring being about half the diameter on the ster- 

 ile coverslip. When this is cool, place 2 drops of sterile culture medium in 

 the center of the ring (on the coverslip) and then add the excised explant to 

 the medium. Place a drop or two of sterile distilled water in the depression 

 slide and a ring of vaseline or petroleum jelly around the margins of the de- 

 pression. Ouickly invert the coverslip and place it over the depression, 

 thereby providing the explant with an air-tight moist chamber. In some in- 

 stances, it will be better to omit the distilled water in the depression, and 

 to use more culture medium and turn the slide (and attached coverslip) up- 

 side down so as to bring the explant against the coverslip to which it will be- 

 come adherent within a day or so. The depression slide may then be re-in- 

 verted, and the explant examined directly (through the coverslip) within the 

 hanging drop of culture nnedium under a dissection or regular microscope. 



Urodele explants should be kept at temperatures of 1 to 1 5°C. and Anuran 

 explants will do better at 1 5 to 18 C. 



OBSERVATIONS AND EXPERIMENTAL DATA: 



Avoid any unnecessary disturbance of the cultures during the first 24 hours. During sub- 

 sequent examinations avoid drastic changes in temperature as from the microscope lamp. 



It is obvious that the radical change to which the organ anlagen are subjected in this type 

 of experiment make it imperative that the material be observed at frequent intervals. In 

 the space below: 



1. Compare the changes in the explant with corresponding changes in the same area 

 of unoperated animals of the same age, and with the control explants. 



2. Make day-to-day sketches of observable changes under the microscope. 



3. If the explant seems to be healthy, attempt to renew the culture medium after 3 to 

 4 days, and continue it as long as possible. (Remember Carrel's chick heart 

 fibroblasts which lasted over 25 years!). 



4. At the conclusion of the experiment, section, stain, and study the differentiation. 



♦ Or white vaseline. 



*♦ Seamless cellophane tubing of small diameter is available in 100 foot rolls and can be cut and tied off into short culture tubes 

 which con be immersed in any constant temperature bath. If about 25% of the space is air, the culture will survive for several 

 days before a change is necessary. 



