30. OSMO-REGULATION 



PURPOSE: To determine: 



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



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



3. The ability of the various stages of development to adjust to anisotonic con- 

 ditions. 



MATERIALS: 



Biological : Ovulating female and nnature 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 vol- 

 ume 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 : 



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

 the control medium. 



2. 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) 



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



4. Standard Solution (Holtfreter ' s) equal to 0. 385% salt, isotonic. 



5. Standard X ij, hypertonic. Equal to 0. 57% salt. 



6. Frog Ringer's, equal to 0.72% 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 em- 

 bryos 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 membranes only. 



2. Unfertilized uterine eggs possessing vitelline membranes, jelly, and all being in 

 metaphase of the second maturation division. These may be transferred from 

 the opened uterus to the appropriate solution by means of a wide-mouthed pipette 

 moistened with Nujol. 



3. Fertilized eggs, transferred to the experimental media 30 minutes after insem- 

 ination. This is about l^ to 2 hours before the first cleavage. 



4. Early cleavage: 4 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 neurulation - medullary plate stage. 



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