50 



CULTURING OF AMPHIBIAN EMBRYOS 



AMPHIBIAN RINGER'S SOLUTION: 



This solution is hypertonic to Standard Solution and to embryos and embryonic tissues, 

 but is satisfactory for adult tissues of the Amphibia. 



NaCl 

 KCl . 

 CaCl- 



0. 66 grams 



0.015 



0. 015 



NaHC03 (Buffer) 0.030 



Distilled water 100. cc. 



(amount necessary to 

 regulate pH at 7. 8) 



ORIGINAL FROG RINGER'S: (S. Ringer 1880: Jour. 



NaCl 



KCl 



Physiol. 13:380) 



0. 65 grams 

 0. 014 



CaCl2 0. 012 



NaHCO-5 



0. 02 



NaH2P04 0. 001 



Glucose 0. 20 



Water to 100. 



SPRING WATER: 



Great Bear Spring Water. This has proven to be entirely satisfactory for the early de- 

 velopment of Anuran embryos particularly. Its chemical analysis is available from the 

 dispensing company in New York City. 



URODELE STOCK SOLUTION: 



The same constituent salts are found in the Urodele media but in slightly different pro- 

 portions. This stock solution could be made up in 20 liter carboys and could be diluted 

 for either operating or growing media, using the Great Bear Spring Water for dilutions. 



NaCl 70. grams 



KCl 1.0 



CaCl2 2. 



Spring Water 10. liters 



URODELE OPERATING MEDIUM: 



This medium is slightly hypertonic to the growing medium. Embryos may be left in this 

 medium for several hours after transplantations, etc. , and then should be transferred to 

 the Growing Solution. 



Urodele Stock Solution 2 parts 



Great Bear Spring Water ... 1 part 



URODELE GROWING SOLUTION: Urodele larvae grow very satisfactorily in this- medium. 



Urodele Stock Solution 1 part 



Great Bear Spring Water ... 4 parts 



In making up synthetic media it is recommended that glass distilled water be used where 

 it is practicable. It has been found that with copper stills, enough copper goes into solu- 

 tion, in some stills, to make the water slightly toxic to embryos. In any crucial experi- 

 ment the investigator should pre-test the medium against the embryos to be studied to 

 that he can eliminate this as a possible factor in his experimental results. The data from 

 the Osmo-Regulation experiments will be of value iii this regard. 



