240 OSMO-REGULATION 



PROCEDURE WITH URODELE EGGS 



Salamander eggs and ennbryos are not generally available in abundance as Anuran 

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

 tities. Such embryos as are available may be segregated into stages and if there are 

 sufficient numbers, some may be deprived of their jelly and vitelline membranes. So far 

 as possible test the covered and nude eggs against the same media, using identical and 

 sufficient numbers in each medium so that the results may be considered statistically 

 valid. It is of utmost importance that all conditions of relative volume of medium per egg, 

 temperature, etc. are identical. 



EXPERIMENTAL PROCEDURE: 



Prepare 2 finger bowls for each of the media, each of which should receive 50 cc. of the 

 solution to be tested. Place 25 eggs or embryos in each finger bowl, all of the same 

 stage of development. The ratio must be 2 cc. of medium per egg or embryo. With 

 Urodele material, which is less abundant, the volume of medium need not be reduced but 

 the number of embryos per container may be less than 25 but must remain the same for 

 all. The environmental conditions of temperature, light, etc. must be identical for all 

 so that the only variable is the n-iediuin being tested against embryonic development. 



Eggs and embryos that are to be deprived of their jelly and vitelline membranes should 

 first be placed in the test medium and then denuded. 



OBSERVATIONS AND EXPERIMENTAL DATA: 



There are three sets of observations to be made : 



1. The survival of eggs or embryos allowed to remain in the various media, begin- 

 ning at different stages of development. These data will have significance not 

 only in relation to isotonicity but also to tolerance, at the various embryonic 

 stages. Duration of observation: one week. 



2. The developmental rate in anisotonic solutions as compared with the controls. 

 These data will be significant only under the condition that the temperature is 

 very accurately controlled. 



3. The diameter readings, or swelling and shrinking of the eggs and embryos. 

 These readings can be taken directly by placing fine-lined graph paper beneath 

 the finger bowl containing the eggs and determining the relative values against 

 the graph paper. Readings should be taken along two axes, at right angles to 

 each other, since some eggs may be ovate. The average of the two readings 

 would then be taken as the value for any particular egg. 



This type of observation has relative value only, and can be used on body cavity 

 eggs, aged eggs, and early cleavages up to but not including the gastrula stage. 

 Gastrulation involves the development of internal cavities which would render 

 these readings invalid. 



If time permits, two additional sets of observations should be made : 



4. The aged uterine egg of the Anuran shows cortical breakdown, beginning after 

 about 5 days at 10 C. , and earlier at the higher temperatures. Such an egg 

 should react more with increasing age toward anisotonic media, eventually re- 

 sponding like any non-living osmometer. Readings may be made over rather 

 short intervals of time. 



5. The tolerance of the extremes in osmotic pressure by the various critical em- 

 bryonic stages exposed for brief periods can be tested. For instance, recently 

 inseminated eggs or eggs about to gastrulate might be exposed for a limited time 

 to glass distilled water, or to double Standard Solution, or to both in succession 

 before returning them to the control medium. 



