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 be- 

 tween the embryonic gut and the belly ectoderm. The most Important single factor for sur- 

 vival 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 minimum the environ- 

 mental 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, labora- 

 tory 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- 

 Eegulation) . The tap water, in large cities particularly, may be so highly chlorinated 

 that It is toxic 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 abun- 

 dant 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, Valeclnerla, 

 Saglttaria, Nltella, 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 par- 

 ticular forms. Some of these formulae follow: 



STANDARD (HOLTFRETEB'S) SOLUTION : 



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

 total salt content is 0.585^, 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 0.55 grams 



KCL . 005 " 



CaClg 0.01 



NaHCO, (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.) 



AMPHIBIAN RINGEB'S SOLUTION : 



This solution is hypertonic to Standard Solution and to embryos and embryonic tis- 

 sues, but is satisfactory for adult tissues of the Amphibia. 



NaCl . 66 grams 



KCL 0.015 " 



CaCl2 0.015 " 



NaHCO^ (Buffer) O.050 " (amount necessary to 



Distilled water 100.0 cc. regulate pH at 7.8) 



- ka. 



