194 PRODUCTION OF DOUBLE EMBRYOS 



remain inverted, or were distorted by the jelly-tension, it would be well to 

 make a sketch record in order to have a possible explanation of later develop- 

 mental monstrosities. 



The original method of placing the eggs between glass plates (glass slides) and 

 compressing them sufficiently to prevent rotation when the plates are inverted, 

 can be attempted. The objection to this method is simply that the pressure fac- 

 tor is not uniform and should be taken into consideration. 



The first cleavage normally occurs 2 hours after the eggs are inseminated, and 

 the second cleavage follows within 1 hour. It is important that the eggs be in- 

 verted immediately after the completion of the first cleavage and not later. It 

 would be well, therefore, to segregate eggs inverted at various stages of the 

 first cleavage development to determine the effect of this variable on double 

 monster production. 



B. DOUBLE EMBRYOS BY CONSTRICTION 



PURPOSE: To determine the ability of single blastomeres of the Z-cell stage to develop 

 complete embryos, following accentuation of the first cleavage furrow. 



MATERIALS: 



Biological: Urodele eggs in the 2-cell stage. Amblystoma eggs may be collected 

 in nature (see section on Breeding Habits) or Triturus eggs may be 

 layed in the laboratory as a result of anterior pituitary stimulation 

 (see Induced Ovulation). 



Technical : Standard Solution for Anura and Growing Medium for Urodela. 

 0. 1% KOH in appropriate medium. 

 Hair loops, silk fibers, operating glass needles. 



METHOD: 



Precautions : 



1. Avoid over-exposure to the KOH solution (see section on Isolation of Embry- 

 onic Cells). 



2. After separating the blastomeres, keep specimen in adequate medium and at 

 a cool temperature. 



Controls: These will consist simply of eggs from the same clutch, kept under iden- 

 tical conditions except for the separation of the blastomeres. 



Procedure : 



Prepare simple loops of fine (blonde) baby's hair so that each loop is slightly 

 greater than the diameter of the egg and its jelly capsule. Prepare several 

 Syracuse dishes with Permoplast base and depressions calculated to hold the 2- 

 cell stage and its jelly capsule. Fill with appropriate medium and then select 

 eggs in the two cell stage for constriction. 



By placing these eggs for a brief period in 0. 1% KOH (made up in the same cul- 

 ture medium) the surface coat will be weakened and the cleavage furrow will be 

 accentuated. Remove the egg and pass it through three changes of culture me- 

 dium before the furrow has progressed very far. This should be practiced, for 

 it may not be easy to stop the KOH action as abruptly as desired. 



Remove the 2-cell stage to the Syracuse dish with Permoplast depression and 

 press the hair loop into the bottom of the depression (two ends above the depres- 

 sion) and maneuver the egg into the depression and loop so that the cleavage 

 furrow lies directly parallel to the loop. With practice one can determine whether 



