16 EXPERIMENTAL EQUIPMENT AND PROCEDURES 



The Niu-Twitty Solution - Three solutions made separately in hot water, cooled, and 

 then mixed. Good lor urodele embryos and isolates. 



Solution A Solution B Solution C 



3, 400 mg. NaCl 110 mg. Na2HP04 200 mg. NaHC03 



50 mg. KCl 20 mg. KH2PO4 250 cc. dist. water 



80 mg. Ca(N03)-H20 250 cc. dist. water 



100 mg. MgS04 

 500 cc. dist. water 



7. Locke's solution - tor chick embryos: 



NaCl 0. 9 gr. 



KCl 0. 04 gr. 



CaCl2 0. 024 gr. (anhydrous) 



NaHC03 0. 02 gr. 



Water 100. cc. 



8. Special variations of the above solutions: 



a. Calcium-tree Standard (Holtlreter ' s) Solution. 



b. Sodium-tree Standard (Holtfreter ' s) Solution. 



c. Potassium-free Standard (Holtfreter ' s) Solution. 



d. Buffer-free Standard (Holtfreter ' s) Solution. 



e. Nuclear medium for germinal vesicle studies (Calcium-free Ringer's). 



NaCl 0. 6 gr. 



KCl 0. 01 gr. 



Glass Distilled Water 100. 00 cc. 



9. Anesthetics : (See also page 20) 



a. MS-222 (Sandoz Chem. Co. , 63 Van Dam St. , N. Y. C. ) Excellent for embryos. 



Make up 1/3000 concentration in Spring Water and in Standard Solution of the 

 above concentrations and keep refrigerated. (Tricain Methanesulfonate) Must 

 be used freshly diluted. 



b. Chloretone - 1/3000 in Spring Water or in Holtfreter ' s. 



c. Ether - anesthetic form. 



d. Chloroform. 



e. Magnesium sulphate (Epsom salts), crystal form. 



1 0. Killing and fixing solutions : 



a. Smith's fixative - best for yolk-laden amphibian eggs. Two solutions to be mixed 

 just before use. Not to be used if discolored. 



Solution A : K Bichromate . . 0. 5 gr. 



Water 87. 5 cc. 



Solution B : Formalin 10 cc. 



Acetic (glacial) . 2. 5 cc. 



b. Bouin' s fixative - safest of all fixatives, particularly for late stages. 



Saturated (aqueous) picric acid. . 75 cc. 



Formalin (commercial) 25 cc. 



Glacial acetic acid 5 cc. 



(Can use 95% alcohol instead of water for more rapid fixation) 



c. Bouin-Dioxan - excellent for yolk embryos and rapid technique. (See Puckett, 

 1937 - Stain Tech. 12:97). Use Bouin's and Dioxan in equal parts (Caution: 

 Dioxan is volatile and toxic). 



d. Michaelis' fluid - for yolk embryos. 



Cone. H CI2 - aqueous 20 pts. 



Cone, picric aqueous 20 pts. 



Glacial acetic acid 1 pt. 



Water distilled 40 pts. 



