44 BASIC METHODS FOR EXPERIMENTS 



separation as the result of Insemination or of treatment with ah 

 organic acid. The membranes are most readily removed imme- 

 diately after separation. This removal is generally accom- 

 plished by shaking. But there is much evidence to indicate that 

 at this time the eggs are very susceptible to shaking. Boveri 

 and others used shaking for the specific purpose of modifying 

 the development of echinid eggs. I should say that any inter- 

 pretation concerning normal development based on the study 

 of eggs whose membranes have been removed by shaking the 

 eggs immediately after membrane-separation is without value. 

 Shaking at this time however is an excellent experimental 

 method for the modification of the normal developmental process. 



The membranes can, however, be removed without the 

 slightest injury to the eggs. For this purpose again I use bolting 

 silk. The method is as follows: 



I use eggs from one female known by previous trial insemina- 

 tions to be of optimum fertilization capacity, as revealed by the 

 speed and quality of the ectoplasmic reactions induced by 

 insemination. About two minutes after insemination when the 

 membranes are equidistant from the egg surface at all points — 

 i.e., when the perivitelline space is of equal width throughout — 

 the &g^ suspension is very gently poured on to the wetted bolting 

 silk. As the eggs pass through the mesh, they lose their mem- 

 branes. Practically lOO per cent of the eggs will thus be freed 

 of their membranes. Eggs without membranes should never be 

 crowded; they should lie in one layer well spaced in a large volume 

 of sea-water. 



Methods for Inducing Membrane-Separation in 

 Unfertilized Echinid Eggs 



In 1893 Herbst repeated and confirmed the observation of the 

 Hertwig brothers that chloroform induces membrane-separation 

 in the sea-urchin egg. He also found that benzol, toluene, 

 xylene, creosote and clove-oil have the same effect. 



Schiicking found that eggs exposed to distilled water separate 

 membranes. This observation has been confirmed by Glaser 

 and by myself. Hypertonic sea-water will also induce mem- 

 brane-separation, I find, in several species of echinid eggs. The 

 most popular method for bringing about the separation of the 



