ON THE EXPERIMENTAL HYBRIDIZATION OF ECHINOIDS. 273 



cases did we observe segmentation in the unfertilized controls, and in these cases the 

 corresponding experiments were discontinued. 



Fertilization was brought about by adding one or two drops of the suspension of 

 sperm to the bowl containing the eggs. During the period of segmentation the eggs 

 were thoroughly washed, with several changes of " outside" water, to prevent any 

 excess of sperm from decaying and subsequently contaminating the culture. 



As soon as the blastulae swam up to the surface of the water in the bowls, a number 

 were pipetted off into jars of about 2 litres capacity, which had previously been washed 

 with hot water, and then sterilized with dry heat. In the majority of cases the 

 larvse were reared in " Berkefeld " water. In some cases the larvae were raised in 

 " outside " Avater. In general the latter method was found less satisfactory than the 

 former, because excessive growth of plankton diatoms was apt to appear, and 

 destroy the larvae. 



From each fertilization, several hundreds of blastulse were pipetted into each of 

 three or four jars of " Berkefeld " water. To each jar was added a small quantity of 

 a pure culture of tlie diatom Nitzschia closterium forma minutissima. The growth 

 of the diatom varies with the temperature. Usually one pipetteful was added to 

 each culture jar. In the hot summer of 1911, however, if more than half a pipetteful 

 was added an overgrowth resulted, which choked the plutei. On the other hand, 

 in the colder weather of spring, about two pipettefuls was necessary. 



On this diatom the young plutei feed with avidity and grow rapidly. These 

 cultures of Nitzschia were obtained in almost a pure state by fractional inoculation 

 into jars of " Berkefeld " water. When once obtained they can be kept constantly 

 growing by fresh inoculation from time to time. We have made use of the cultures 

 originally obtained by Allen and Nelson (3) and which have been in continuous 

 growth for four or five years. With care these can be kept almost free from ordinary 

 bacterial contamination. 



The jars were examined from time to time to see that no undue growth of diatoms 

 took place, that would be likely to injure the larvse. If this occurred, the larvae 

 were pipetted out into fresh culture jars, the temperature of the jars varying from 

 10° to 16^ C. 



When the Echinus-rudiment had grown to a comparatively large size, the plutei, 

 which had hitherto been swimming at or near the surface of the water, sank to the 

 bottom of the jars. Here they metamorphosed, and as soon as this occurred, they 

 were removed to fresh dishes, for if allowed to remain in the original jar they soon 

 became choked by the deposit of Nitzschia on the bottom. 



In the past we did not know the correct food for the young Echini after meta- 

 morphosis, and they were just placed in dishes, in the hope that they would find 

 their right food among the growths that appeared there. During the summer of 

 1912, however, we have discovered a method of feeding the urchins, so that they 

 grow rapidly. 



2 N 2 



