FERTILIZATION REACTION IN ECHINARACHNIUS PARMA. 43 



the experiments cited above the butyric mixture previously pre- 

 pared was added directly to the Syracuse glass containing the 

 eggs. I was content to work with relatively a small number of 

 eggs always from but one female. 1 The amount of butyric mixture 

 used, therefore, could be likewise small (in my experiments 

 never more than 12.5 c.c.) so that the eggs could be thoroughly 

 stirred up and still settle quickly with the result that when at 

 most the I to 1.5 c.c. was removed the amount of acid carried 

 over was negligible. 2 



i. Effect of Washing. Dry eggs may be washed quickly once 

 or twice in a small amount of sea-water without affecting the 

 butyric treatment. Additional washings will cut down the 

 per cent, of butyric membranes and washings extending over 

 one to two hours will result in the failure of butyric acid to form 

 membranes. Eggs similarly treated may be still capable of 

 fertilization. Eggs incapable of fertilization never respond to 

 butyric acid treatment. . 



An experiment of July 26 shows the difference between dry 

 and washed eggs : 



8:55 A.M. A fine lot of eggs divided among three dishes 

 A, B and C. Lot A dry; Lot B washed three times in fifty 

 minutes; to lot C two drops of body fluid added. 



10.00 A.M. Lot B gave the following results after exposure 

 to butyric sea- water: 



Exposure in seconds 20 25 30 35 40 60 



Per cent, of membranes 6 19 38 27 14 i 



Lot A (unwashed) per cent, of membranes 18 36 76 91 83 i 



On August 4 similar results were obtained. After four wash- 

 ings in thirty minutes the per cent, of membranes formed was 

 reduced one half after optimum exposure as compared with the 

 unwashed. 



1 Indeed, in my judgment the practise of using large quantities of eggs from a 

 number of females is a most pernicious one, introducing as it does more unknown 

 factors. Nothing is so variable as the echinid egg, as Tennent, Medes and Gold- 

 farb have shown the work of the last-named being particularly concerned with 

 our problem; in a lot of eggs from several females one must get a very heterogeneous 

 population especially, as is usually the case, if the worker obtains his eggs by 

 mincing the ovaries in sea-water. 



- Transfers were sometimes made to 4,000 c.c. of normal sea-water; the results 

 obtained were no better than those to 250 c.c. of sea-water. 



