392 



EXPERIMENTAL FISH EMBRYOLOGY 



2. Culture medium: For marine forms the filtered and sterilized sea water can 

 be used. For fresh-water forms, Standard (Holtfreter' s) Solution should be 

 used, in normal and in double strength. To vary the medium, it is suggested 

 that 0. 1% glucose or 1% glucose plus 0. 1% peptone be added prior to auto- 

 claving. (See various media used with chick explants. ) 



3. Pass the egg to be studied through 7 to 10 changes of sterile medium iso- 

 tonic for that egg, to free it of most of the bacteria. 



4. Decapsulate the egg in the manner described above (Nicholas, 1927) at stages 

 #2 to #7. 



5. Using sharp, sterile, steel knives dissect away the blastodermic disc of any 

 stage from 1 to 32-cells. Clean away all adherent yolk granules and culture 

 in hanging drop (see chick exercise), in sterile medium. (These will gener- 

 ally form hyperblastulae. ) 



6. From later stages (stages #7 to #12) explant the entire embryonic mass (ex- 

 clusive of the yolk) into culture dishes with hypertonic and sterile media 

 and observe for 48 hours or more for differentiations. Histological analyses 

 will be necessary to determine the degree of differentiation. 



7. Bisect the entire egg of stage # 1 3 in the manner indicated in the diagram be- 

 low, using a sharp and sterile steel needle. As the needle is pressed through 

 the egg and into the underlying Permoplast base of the operating dish, the 



cut surfaces of the two halves are pinched apart in such a manner that the 

 cut surfaces are usually closed together and the yolk is contained within the 

 half-sized vesicles. When this occurs, the entire halves may be cultured. 

 When there is rupture, parts of the germ ring and embryonic shield should 

 be further dissected away and cultured in isolation. 



The localization of presumptive nervous system, 

 mesoderm and endoderm in the early gastrula (A) 

 and the middle gastrula (B), and the position of 

 these tissues in the seven-somite embryo (C). 



The position of the nervous tissue is indicated by 

 heavy stipple (forebroin and optic vesicles), di- 

 agonal hatching (mid-brain and anterior hind- 

 brain) and vertical hatching (posterior hind-brain 

 and spinal cord). The cells whose position is in- 

 dicated by horizontal hatching aid in the forma- 

 tion of mid-brain, hind-brain and spinal cord. 

 The mesoderm is indicated by the lightly stippled 

 areas. The areas marked by the numbers 1, 2, 3 

 and 4 ultimately lie in the regions of the embryo 

 indicated by the arrows accompanying diagram C. 



The endoderm is represented by open circles. The 

 endodermal cells that have invaginated are not 

 shown. 



Scheme of operations. In order to isolate 

 the germ ring of late gastrulae originally 

 located 180 away from the dorsal lip of 

 the blastopore, the eggs are divided into 

 two parts by cutting along the plane X-X 

 shown in Fig. lA. Fig. IB shows a dorsal 

 aspect of the blastoderm. The cross- 

 hatched regions represent the material in- 

 volved in the grafts of germ ring originally 

 90° or 180° from the embryonic axis. The 

 heavy stippling indicates germ ring (GR) 

 and embryonic shield (ES), the light stippl- 

 ing extra -embryonic membrane. 



(From Oppenheimer 1938: 

 Jour. Exp. Zool. 79:185) 



(From Oppenheimer I93t): 

 Jour. Exp. Zool. 73:405) 



