CULTURING ISOLATED ANLAGEN 



219 



Procedure: 



1. Prepare 2 culture media at least . For Anura use Standard Solution and for 

 Urodela use Growing Medium, each of which should be sterilized by boiling 

 or autoclaving. Boiling must be brief to avoid changing the salt concentra- 

 tion. The second type of culture medium should be either of the above (de- 

 pending upon whether Anura or Urodela are used) to which is added peritoneal 

 fluid. This can be done by injecting about 3 cc. of the salt solution into the 

 body cavity of adults of the same species, preferably females and then re- 

 moving it, with the same hypodermic, after a few minutes in the body cavity. 

 This second medium cannot be boiled, but the 0. 5% sodium sulfadiazine 



should be added. 



2. Prepare the culture dishes: 



a. Place a layer of absorbent cotton on the bottom of a Petri dish, and then 

 mould a place in the center of the cotton for a small watchglass. Place 

 on the cover and sterilize in the autoclave. Allow it to air cool. Just 

 before placing the culture medium and the explant in the watchglass, it 

 will be necessary to moisten the cotton with sterile distilled water. 

 Avoid placing any water in the watchglass, and undue exposure to the 

 bacteria of the air. 



b. Wrap some depression slides individually in white paper, and heat steril- 

 ize (at 150 C. ) for 15 minutes. Circular coverslips may be autoclaved 



in a #2 covered Stender, or sterilized in 95% alcohol. 



3. Prepare the explants: 



The following anlagen make excellent explants: 



Gill - try ectoderm alone, ecto- and mesoderm, and then include the 

 pharyngael endoderm. (See Moser, 1940) 



Limb - try ectoderm alone, then include underlying mesoderm. (See 

 Harrison, 1928) 



Balancer (Urodele) - use both ecto- and mesoderm components. 



Sucker (Anura) - use both ecto- and mesoderm components. 



Olfactory placode - use ectoderm alone. 



Eye - use optic vesicle, with and without overlying ectoderm. (See 

 Filatow, 1926) 



Tail - include both ecto- and mesoderm. 



Heart - remove ventral ectoderm and allow it to wrap itself around some 

 of the heart mesenchyme. (See Stohr, 1924) 



Neural crest - peel off the dorsal ectoderm and isolate the cord and at- 

 tached crest alone (see section on "Neural Crest Origin of Pig- 

 ment"). 



COVER SLIP 



CULTURE 



EXPLANT 



MEDIUM (HANGING DROP) 



MOIST CHAMBER 



STERILE WATER 



SCHEMATIC SECTION THROUGH 

 DEPRESSION SLIDE WITH EXPLANT 



