EXPERIMENTAL FISH EMBRYOLOGY 



U05 



Procedure: 



Insure complete aaepals, autoclaving the media and sterilizing all instruments 

 and glassware. Culturing can be in deep depression slides, or in covered and 

 sealed #1 Stenders. 



Culture medium: For marine forma the filtered and sterilized sea water can be 

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

 in normal and in double strength. To vary the medium, it is suggested that O.l'jt 

 glucose or 15^ glucose plus O.l'jt peptone be added prior to autoclaving. (See vari- 

 ous media used with chick explants.) 



Pass the egg to be studied through 7 to 10 changes of aterlle medium isotonic for 

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



Decapsulate the egg in the manner described above (Nicholas, 1927) at stages #2 

 to #7. 



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

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

 hanging drop (see chick exercise), in sterile medium. (These will generally form 

 hyperblaatulae. ) 



From later stages (stages #7 to #12) explant the entire embryonic mass (exclusive 

 of the yolk) Into culture dishes with hypertonic and sterile media and observe for 

 h8 hours or more for differentiations. Histological analyses will be necessary 

 to determine the degree of differentiation. 



Bisect the entire egg of stage #15 in the manner indicated in the diagram below, 

 using a sharp and sterile steel needle. Aa the needle is pressed through the egg 

 and into the underlying Permoplaat base of the operating dish, the cut surfaces 

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

 usiially closed together and the yolk is contained within the half-aized 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 cul- 

 tured 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 (forebraln and 

 optic vesicles), diagonal hatching (mid- 

 brain and anterior hlnd-braln) and vertical 

 hatching (posterior hlnd-braln and spinal 

 cord). The cells whose position Is Indi- 

 cated by horizontal hatching aid In the 

 formation of mid-brain, hlnd-braln and 

 spinal cord. 



The mesoderm is indicated by the light- 

 ly stippled areas. The areas marked by the 

 numbers 1, 2, 3 and 4 ultimately lie in the 

 regions of the embryo indicated by the ar- 

 rows accompanying diagram C. 



The endoderra Is represented by open 

 circles. The endodermal cells that have 

 invaginated are not shown. 



(From Oppenheimer I956: 

 Jour. Exp. Zool. 7^:kO^) 



Scheme of ope 

 Isolate tlie germ 

 originally locate 

 dorsal lip of tlie 

 are divided into 

 along the plane X 

 Fig. IB shows a d 

 blastoderm. The 

 represent tlie mat 

 grafts of germ rl 

 I8O" from the emb 

 stippling Indicat 

 embryonic shield 

 Ing extra-embryon 



rations. In order to 

 ring of late gastrulae 

 d 18(i" away from the 



blastopore, the eggs 

 two parts by cutting 

 -X shown in Fig. lA. 

 orsal aspect of tlie 

 cross-hatched regions 

 erial involved in the 

 ng originally UO" or 

 ryonlc axis. The heavy 

 es germ ring (OR) and 

 (ES) , the light stlppl- 

 Ic membrane. 



(From Oppenheimer 195S: 

 Jour. Exp. Zool. 79:185) 



