2. THE CULTURE OF AMPHIBIAN EMBRYOS 

 AND LARVAE TO METAMORPHOSIS 



Early amphibian embryos possess an abundance of yolk which provides them with all the 

 nutriment necessary for a considerable period of development. Tadpoles of both the 

 Anura and the Urodela can survive for many days after hatching, by utilizing the yolk 

 found between the embryonic gut and the belly ectoderm. The most important single fac- 

 tor for survival during the earliest stages of development is the temperature, and second 

 to this is the culture medium. In order that the research worker can reduce to a mini- 

 mum the environmental variables, some suggestions regarding the culturing of common 

 amphibian forms are given here. 



CULTURE MEDIUM 



All amphibia lay their eggs in water. The pond water in which the form to be studied is 

 known to breed is the ideal water to use. Since this is not always practical, laboratory 

 substitutes have been devised, based partly upon a chemical analysis of such pond waters. 

 In general it has been found that slightly hypotonic media are preferred, and development 

 can proceed even in distilled water to some extent (see section on Osmo-Regulation). 

 Tap water, in large cities particularly, may be so highly chlorinated that it is tojcic to 

 embryos or, in some instances, enough metallic ions escape from the lead, copper, or 

 iron piping that the embryos cannot survive. Tap water which has been run through sand 

 and charcoal, filtered, and allowed to stand for several days with abundant plant material 

 in it, will generally prove quite satisfactory. The sand and charcoal take out the debris 

 and dissolved gases, and the living plant material (Elodea, Valecineria, Sagittaria, 

 Nitella, etc.) helps to increase the oxygen content. 



It is now quite clear that sodium, calcium, and potassium ions must be present in an 

 approximate ratio of 50:1:1 in order that development of aquatic embryos be normal. 

 Each of these ions has specific value in cleavage and the developmental process, and if 

 the ratio is maintained the forms can tolerate quite a range in concentration. Solely by 

 the empirical method a number of formulae have been devised, each presumably suitable 

 to particular forms. Son^ie of these formulae lollov 



Dw: 



STANDARD (HOLTFRETER'S) SOLUTION: 



This solution has proven to be the niost satisfactory of the synthetic media. The total 

 salt content is 0. 385%, which is hypotonic to adult tissues but seems to be isotonic to the 

 early embryonic stages of the Anura. It is recommended that this solution be made up in 

 double or quadruple strength as a stock solution. The normal concentration follows: 



NaCl 0. 35 grams 



KCl 0. 005 " 



CaCl2 0. 01 



NaHC03 (Buffer) 0.02 



Distilled water 100. 00 cc. 



( Note : This solution is satisfactory for T. pyrrhagaster if the NaCl is reduced to 

 half and the buffer is omitted. ) 



