394 



EXPERIMENTAL FISH EMBRYOLOGY 



If the period ol isolation can be extended to more than 4 days it will be necessary to re- 

 new the culture medium at that time. Generally the limit of differentiation will be 

 achieved before that time, usually within 48 hours. If the culture is made in a hanging 

 drop (see chick exercises) the complete process of development can be observed under 

 the dissecting microscope without disturbing the explant, and if it is bacteria-free, it 

 should survive. 



Yamamoto (1939 Fac. Sci. Tokyo Univ. 15:269) gives a formula for a synthetic medium 

 which is isotonic to the Oryzias egg. 



M/7. 5 NaCl 100 cc. 



M/7. 5 KCl 2. cc. 



M/11 CaCl2 2. 1 cc. 



pH to 7. 3 



To such a medium 0. 1% glucose may be added for nutrition for the explants of Oryzias. 



Hayes, Darcy and Sullivan (1946: Jour. Biol. Chem. 163:621) have analyzed the ovarian 

 or coelomic fluid of the salmon, which fluid appears suddenly just at the time of hatch- 

 ing. They find it to be a clear, limpid, and slightly translucent medium with the follow- 

 ing constituents: 



Ions per 1, 000 liter of water: 



Sodium 151 milliequivalents 



Potassium 3.2 



Calcium 7.1 



Magnesium 2. 6 



Chlorine 116. 



HPO^ 

 HCO, 



4. 

 13. 4 



Since the ripe eggs of the Salmon lie freely within the body cavity, and hence within this 

 fluid medium, this medium may be considered as isotonic to the eggs at this stage. This 

 fluid is, however, hypertonic to blood. Sodium chloride is the dominant salt, with other 

 ions in the approximate ratio that they are found in sea water. In the eggs the potassium 

 dominates, and the calcium and magnesium are not osmotically active. At fertilization, 

 in water, the egg loses osmotic pressure by about 3%. During development the egg and 

 embryo take up calcium and sodium from the environment so that the final amounts are 

 4 and 3 times the initial amounts respectively. Phosphorous intake is markedly in- 

 creased, probably in connection with sketeon formation. (See references by Trinkhaus 

 &t Drake 1956: Devillers 1957) 



d. Induction of Secondary Embryo by Grafting of Dorsal Lip 



The fish egg can be used as a host for successful transplantations without the necessity 

 of a Briicke or bridge to hold the graft in place. Healing and development are so rapid 

 that the whole experiment can be concluded within several days. 



Procedure : 



1. Pass the egg and its chorion through 7 to 1 changes of sterile medium. 

 This should be hypertonic, such as double or triple Standard (Holtfreter ' s) 

 Solution. 



2. Decapsulate the egg, using the technique of Nicholas (1927) described above. 

 The steel needles or watchmaker's forceps must be sterile. Use stage #13 

 when the germ ring has passed over about 3/4 of the yolk. Similarly pre- 

 pare the donor, of the same age and stage. 



