OSMO-REGULATION 



PURPOSE ■• To determine: 



1. The ability of eggs and embryos to tolerate various osmotic conditions. 



2. The ideal concentration (isotonic nedium) for the various stages. 



5. The ability of the various stages of development to adjust to anisotonlc 

 conditions. 



MATKRIALS : 



Biological : Ovulating female and mature male frogs; Urodele embryos. 



Technical: Various salt solutions listed below. 



Fine-lined graph paper mounted on underside of Petri dish. 



METHOD : 



Precautions : 



1. Prevent evaporation from containers by properly covering them during the course 

 of the experiment. If evaporation does occur, replace the lost volxune with 

 glass-distilled water. 



2. Avoid crowding. The number of eggs or embryos must be the same in all of the 

 containers, and should not exceed 25 per finger bowl of 50 cc. fluid. 



Controls : 



a. For frog's eggs and embryos, consider Spring Water or Standard Solution as the 

 control medium. 



b. For Urodele eggs and early embryos, consider Urodele Growing Solution as the 

 control medium. 



Procedure : 



Prepare the following media for use in this experiment: 



1. Distilled water, hypotonic medium. (Glass distilled water if possible) 



2. Half Standard, hypotonic, (50^ Holtfreter's equal to 0.192^ salt) 

 5, Spring Water, isotonic. (Great Bear Spring Water) 



k. Standard Solution (Holtfreter's) equal to 0.585^ salt, isotonic. 



5. Standard X 1^, hypertonic. Equal to 0.571^ salt. 



6. Frog Singer's, equal to 0.72?t salt. 



7. Double Standard hypertonic (2 X Holtfreter's) equal to 0.77^ salt. 



PROCEDURE WITH ANURAN EGGS 



The Anura are particularly suitable for this type of investigation for the eggs and 

 embryos are available in quantity and under controlled conditions. It is recommended that 

 the following stages be tested against the various solutions: 



1. Body cavity eggs possessing vitelline membreines only. 



2. Unfertilized uterine eggs possessing vitelline membranes, Jelly, and all 

 being in metaphaae of the second maturation division. These may be trans- 

 ferred from the opened uterus to the appropriate solution by means of a 

 wide-mouthed pipette moistened with Nujol. 



5. Fertilized eggs, transferred to the experimental media 50 minutes after 



insemination. This is about I5 to 2 hours before the first cleavage. 

 h. Early cleavage: U to 8 cell stages. 



a. Jelly Intact. 



b. Jelly removed (with sharp watchmaker's forceps). 



5. Beginning of gastrulation - slit blastopore stage. 



6. Beginning of neuriilation - medullary plate stage. 



PROCEDURE WITH THE URODELA 



Salamander eggs and embryos are not generally available In the abundance of Anuran 

 eggs, nor can they be secured In identical stages of development in any suitable quan- 



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