46 BASIC METHODS FOR EXPERIMENTS 



For eggs in lo cc. of distilled water, 250 cc. of normal sea-water 

 suffices. 



To induce membrane-separation in hypertonic sea-water, the 

 method depends upon the degree of hypertonicity as well as upon 

 the species of egg. With this method my procedure is as follows : 



Make up 3 solutions of 2}'2M NaCl or KCl plus sea-water in 

 the following proportions: 20, 22 and 24 parts of the salt plus 80, 

 78 and 76 parts respectively of sea-water. Flood with the 

 hypertonic sea-water a drop of thick egg-suspension in a Syracuse 

 watch-glass mounted under low power of the microscope. The 

 moment that the eggs separate their membranes, put them into 

 dishes containing at least 250 cc. of sea-water. After several 

 trials with each solution, use that which gives membrane-separa- 

 tion in the shortest time. The rate of membrane-separation will 

 vary somewhat on eggs of the same species even when these, as 

 learned previously by trial insemination, are in the best physio- 

 logical condition. 



I find that pure 2i'^M NaCl or KCl will bring about mem- 

 brane-separation often in less than i minute. But with this 

 solution, the eggs are induced to complete development. Thus, 

 this is a method not only for membrane-separation, but also for 

 experimental parthenogenesis. 



The butyric acid method employed by Loeb (1913 and earlier) 

 has been extensively used by others. Here again the method 

 varies depending upon the species of egg. The important point 

 is to avoid over-exposure to the butyric acid-sea-water, for eggs 

 over-exposed do not separate full membranes; they are injured 

 without loss of capacity for fertilization. One should also avoid 

 using too great concentration of the acid. The method as modi- 

 fied by Heilbrunn and used subsequently by F. R.. Lillie, C. R. 

 Moore and myself for the egg of Arbacia is applicable, I find, to 

 eggs of other echinids. My use of this method follows: 



Have prepared a cylinder containing 50 cc. of sea-water plus 

 2 cc. )f normal butyric acid. Shake the cylinder vigorously. 

 Add the butyric acid-sea-water to eggs in a flat-bottom glass 

 vessel. At 10 second intervals remove about i cc. of the egg- 

 suspension to 250 cc. of sea-water. In this wise establish a 

 series consisting of 10 to 12 numbers. Examine samples from 

 each dish and estimate the percentage of eggs with fully sepa- 



