i go Phylum Annelida 



REMOVAL OF OUTER MEMBRANE 



The outer membrane of the egg may be removed by following a slight 

 modification of a procedure described by Hatt (1932). Two solutions. 

 A and B, are prepared as follows: 



Solution A. — A given volume of a solution of i gm. of Na 2 CO3 

 in 1000 gm. of isotonic NaCl (or KC1). This gives a solution with 



pH 9.6. 



Solution B. — 0.45 cc. of 1.0 n HC1 are added to 100 cc. of iso- 

 tonic NaCl (or KG). This solution has enough HC1 so that when an 

 equal amount of the solution is added to a given amount of Solution 

 A the resulting mixture is at the pH of seawater (8.2). 



The eggs are placed in a Syracuse dish and as much of the seawater as 

 possible withdrawn, without injury to the eggs. A small amount (three 

 to four cc.) of Solution A is poured on them and the dish rotated. After 

 about one minute, the eggs are allowed to settle and the solution with- 

 drawn. Now a carefully measured amount (five cc.) of Solution A is 

 pipetted into the dish. The eggs clump into large masses, but as the 

 membranes dissolve the eggs separate and the masses break up. The 

 disappearance of the egg membranes may be followed under the micro- 

 scope. When they have disappeared from most of the eggs (about five 

 minutes) five cc. of Solution B is quickly added. The force of the 

 stream from the pipette is sufficient to mix the solutions. After the eggs 

 have again settled, they are transferred to fresh seawater in Syracuse 

 dishes. The bottoms of the dishes have been previously coated with 

 a thin layer of agar to prevent the adhesion of the eggs to the glass 

 surface. 



This treatment removes the outer membrane but the jelly still remains 

 about the egg. 



Bibliography* 



Dehorne, A. 1910a. La division longitudinale des chromosomes dans la sperma- 

 togenese de Sabellaria spinulosa. Compt. Rend. Acad. Sci. 150:1195. 



1910b. Le valeur des anses pachytenes et le mecanisme de la reduction chez 



Sabellaria spinulosa. Ibid. 150:1625. 



191 1. Recherches sur la division de la cellule. II. Homeotypie et Hetero- 



typic chez les Annelides polychaetes et les Trematodes. Arch, de Zool. Exp. S. 

 9,1:1. 



1 913. Nouvelles recherches sur les mitoses de maturation de Sabellaria spin- 



ulosa. Compt. Rend. Acad. Sci. 156:485. 

 Faure-Fremiet, E. 1924. L'oeuf de Sabellaria alveolata L. Arch. d'Anat. Micr. 

 20:211. 



*The literature on the European species of this genus is included in the bibliography. I 

 will shortly publish accounts of (i) the details of fertilization, (2) the later cleavage, 

 and (3) the results of experiments with centrifugation and isolation and transplantation of 

 blastomeres. 



