24 ' FRANK R. LILLIE. 



washings, they still continue to produce the agglutinating sub- 

 stance in sea-water, though in much diminished quantity; in my 

 full paper, which Loeb had not the opportunity of consulting, 

 I gave series of measurements on this point (1914, pp. 532-538) ; 

 I also pointed out that in immature ovaries containing many 

 primary ovocytes, but some mature eggs, the quantity of agglu- 

 tinating substance produced was relatively very small (191 4, 

 p. 530), and I therefore suggested that the substance was secreted 

 by the eggs at the time of maturation and was soaked up by the 

 jelly as by a sponge. The eggs, however, continue to produce 

 it after maturation, as I shall show. The immature eggs have as 

 thick a chorion as the mature eggs; therefore the agglutinating 

 substance cannot be merely dissolved chorion. I recognized the 

 possibility of the view expressed by Loeb, investigated it as fully 

 as possible at the time, and rejected it. 



Since Loeb's paper has appeared, I have repeated his experi- 

 ments and found my former observations and conclusions con- 

 firmed in all respects: 



Experiments. — The optimum concentration of HCl for removal 

 of jelly without injury to eggs was found to be 50 c.c. sea-water 

 -\- 1.4 c.c. N/ioHCl. 1.2 c.c. N/io HCl in 50 c.c. sea-water did not 

 fully remove the jelly, and 1.6 c.c. caused too much injury to 

 the eggs evidenced by heavy agglutination and later cytolysis. 

 In an experiment of July 17, 1914, the three above concentrations 

 were used. The complete removal of the chorion in the inter- 

 mediate concentration was demonstrated by observation of the 

 eggs in a thick suspension of India ink in sea- water; even the 

 minutest traces of adherent jelly can readily be detected by this 

 method, but it was all gone. The eggs were then washed as 

 follows: 10. 1 1 A.M. 42/6 c.c; 10.40 51/5 c.c; 10.58 50/4 c.c 

 The supernatant fluid was then tested and found to be free from 

 sperm agglutinating substance; thus furnishing proof that all 

 originally contained in the jelly had been \^ashed out. At 11.20 

 the supernatant fluid was poured off leaving only 5 c.c in the 

 tube. The eggs were allowed to settle, and at 11.25 the super- 

 natant fluid was tested and gave a 9-10-second agglutination 

 reaction with fresh sperm suspension. Thus these eggs entirely 

 deprived of jelly by HCl are producing agglutinating substance. 



