14 E. E. JUST. 



of milky sperm suspension to two cubic centimeters of strong 

 egg-water. 1 The sperm form clumps of such size that they are 

 clearly visible to the naked eye, the sea-water between the masses 

 remaining clear, so sharp is the reaction. This method suffices 

 merely to indicate the fact of agglutination, and this only in high 

 concentration. It was employed but seldom in these experi- 

 ments as will be noted in the following pages. Indeed, by this 

 method alone the critical points in this whole work would scarcely 

 have been determined. 



The methods for handling eggs and sperm are important since 

 the state of the egg or sperm suspension conditions the experi- 

 ment. To obtain eggs relatively free of debris, a mass of ovaries 

 in two or three times their bulk of sea-water is strained through 

 cheese cloth. Or, the animals may be rapidly opened and placed 

 aboral side down in dry watch glasses and dry eggs collected as 

 they exude from the genital pores. Also, eggs may be got as 

 they are normally shed by animals in separate glasses. Sperm 

 is collected from those males that have been opened, dried and 

 placed aboral side down in dry watch glasses, and that soon shed 

 sperm being pipetted off as needed. The sperm suspensions 

 mentioned below, unless specifically noted to the contrary, were 

 always one per cent, suspensions made by the addition of one drop 

 of dry sperm to ninety-nine drops of sea-water. It is imperative 

 to make up the suspension only as needed, for sperm reactions 

 show wide variations depending upon freshness and upon con- 

 centration which likewise is a factor in ageing; the less concen- 

 trated suspensions deteriorate faster than more concentrated. 

 Finally, everything was kept scrupulously clean, and all means 

 taken to prevent contamination which might easily have in- 

 validated whole series of experiments. 



(a) Variations in Fertilizin Production. Eggs vary in their 

 capacity for producing fertilizin. In part this variation may 

 be due to the fact that some suspensions do not consist wholly of 

 eggs but may contain bits of tissue, etc.; such a suspension will 

 be inferior to another of equal concentration but containing eggs 

 only. However, this does not fully explain the variation, for it 



1 By egg water is meant sea-water taken from above eggs that have stood for a 

 time. 



