EXPERIMENTAL EQUIPMENT AND PROCEDURES IS 



SOLUTIONS AND REAGENTS 



1. Distilled water - glass distilled preferred. Supply in large carboys with siphon and 

 pinch clamp. 



2. Spring water - if possible supply in carboys with siphon and pinch clamp. Great Bear 

 Spring Water (N. Y. City) is excellent. 



3. Conditioned tap water - to be stored in carboys after conditioning. This will be 

 necessary where the City water supply is treated so that embryos cannot survive in 

 it. This may be tested with sperm or early embryos. Conditioning is achieved by 

 running tap water into large tank in which there will be maximum of surface exposed 

 and in which plant material is abundant. Artificial aeration will help to eliminate 

 chlorine. Passing tap water through fine gravel and charcoal is rarely necessary 

 but would aid in conditioning highly toxic water. Three or four days of such condi- 

 tioning should be sufficient. 



4. Standard (Holtfreter ' s) Solution - this should be available to the students in several 

 concentrations and large volumes. For the convenience of the instructor, the dry 

 salts may be made up in appropriate concentrations and stored in vials to be added 

 to carboys of glass distilled water when needed. The concentrations needed are 

 200%, 100% and 20%. The formula for Holtfreter' s solution (J. Holtfreter, 1931 - 

 Arch. f. Ent. mech. 124:404) is: 



NaCl 0. 35 gr. 



KCl 0. 005 gr. 



CaCl2 0. 01 gr. 



NaHC03 0. 02 gr. 



Water 100. 00 cc. (glass distilled preferred) 



5. Amphibian Ringer's - should be available in concentrated form: 



NaCl 0. 66 gr. 



KCl 0. 015 gr. 



CaCl2 0. 015 gr. 



NaHC03 to pH 7. 8 



Water 100. cc. 



6. Urodele stock solution : 



Great Bear Spring Water ... 10 liters 



NaCl 70 grams 



KCl 1 gram 



CaCl2 2 grams 



Urodele Operating Medium: (hypertonic) 



Stock solution 2 parts 



Great Bear Spring Water ... 1 part 



Urodele Growing Medium: (isotonic) 



Stock solution 2 parts 



Great Bear Spring Water ... 4 parts 



Steinberg medium: - solution made up complete and autoclaved. Essential to use 

 glass distilled water and can be used for raising whole embryos. (pH 7. 4) 



20 cc. of 17% NaCl 

 10 cc. of 0. 5% HCl 

 10 cc. of 0. 8% Ca(N03)- 4H2O 

 10 cc. of 2. 05% MgS04- 7H2O 

 4 cc. of 1% N.HCl 

 560 mg. of Tris* 

 946 cc. glass distilled water 



* Buffer available from Sigma Chem. Co. , St. Louis. 



