250 



CULTURING ISOLATED ANLAGEN 



An alternative method* ia to prescrite a small ring with a sterile glass rod 

 dipped into soft paraffin**the ring being about half the diameter on the sterile 

 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 depression. 

 Quickly invert the coverslip and place it over the depression, thereby providing 

 the explant with an air-tight moist chamber. In some Instances, 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) upside down so as to bring the 

 explant against the coverslip to which It will become adherent within a day or 

 so. The depression slide may then be re-inverted, and the explant examined di- 

 rectly (through the coverslip) within the hanging drop of culture medium under a 

 dissection or regular microscope. 



Urodele explants should be kept at temperatures of 10° to 15°C. 

 explants will do better at 15° to 18°C, 



OBSERVATIONS AND EXPEBIMENTAL DATA: 



and Anuran 



Avoid any unnecessary disturbance of the cultures during the first 2h hours. During 

 subsequent 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 in- 

 tervals. In the space below: 



a. 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. 



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



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

 5 to *+ days, and continue it as long as possible. (Remember Carrel's chick 

 heart fibroblasts which lasted over 25 years'.). 



d. At the conclusion of the experiment, section, stain, and study the differentia- 

 tion. 



W 



IP 



9 



Progressive development of gill 

 anlagen of AmDljstoma in isola- 

 tion. 



From Moeer, I9I+O: 

 Jour. Morph. 66:26l 



*0r 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 can be immersed in any constant tempera- 

 ture bath. If about 25^ of the space is air, the culture will survive for several days 

 before a change is necessary. 



