CULTURING ISOLATED ANLA6EN 



229 



Prepare the culture dishes: 



a. Place a layer of atsortent cotton on the 'bottoni 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 

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

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

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

 teria of the air. 



"b. Wrap some depression slides individually in white paper, and heat sterilize 

 (at 150°C.) for 15 minutes. Circular coverslips may be autoclaved in a 

 #2 covered Stender, or sterilized in 95^ alcohol. 



Prepare the explants : 



The following anlagen make excellent explants: 



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



pharyngael endoderm. (See Moser, 19^+0) 

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

 son, 1928) 

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

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

 Olfactory placode - use ectoderm alone. 

 'Siye - 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, 192'*-) 

 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") . 

 (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 included in 

 each explant. ) 



COVER SLIP 



CULTURE MEDIUM (HANGING DROP) 

 EXPLANT 



PARAFFIN SEAL 



MOIST CHAMBER 



STERILE WATER 



SCHEMATIC SECTION THROUGH 

 DEPRESSION SLIDE WITH EXPLANT 



The embryo is first passed through several changes of sterile culture medium 

 to remove adherent bacteria, and then the anlagen are removed by means of Iridec- 

 tontr scissors, lancets, glass needles, etc. and are transferred to the culture 

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



h. 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 chamber when 

 the Petri dish cover is replaced. 



