DEFECTIVE PLUTEUS LARV/E. 381 



ior fifteen or twenty minutes, gradually flattening against each 

 other as the next division approaches, until at the time of the 

 second cleavage they appear as half spheres with their flattened 

 diameters in contact. The same process occurs at each cleavage, 

 and is apparently caused by the gradual recurrent increase in 

 viscosity up to the time of actual division. The blastomeres 

 must be separated during the earlier period when they are 

 sphere shaped, since it becomes increasingly difficult later. 



I have made many attempts to separate the blastomeres of 

 Arbacia by the use of Ca-free sea water, so widely used for this 

 purpose since the original discovery of the method by Herbst 

 but all of these have been unsuccessful. The original Herbst 

 solution used at Naples, sea water treated with Na-citrate, 

 and various artificial sea waters without Ca, have uniformly 

 failed to bring about the separation of Arbacia blastomeres 

 without shaking, and this makes it impossible to keep the two 

 blastomeres of one egg under observation. With Echinarachnius 

 the method seems to be somewhat more successful, but not 

 uniformly enough to be superior to mechanical separation. 



The isolated blastomeres were picked up one by one with a 

 capillary lip pipette, and the pairs from any one egg placed to- 

 gether in sea water in a round bottomed glass dish two or three 

 centimeters in diameter with a slot ground in the center (Lefevre 

 dishes). These dishes were more satisfactory than depression 

 slides or other containers, since by placing the blastomeres in the 

 slot the water can be changed without danger of sucking up the 

 cells. The development was observed under the four millimeter 

 objective of the compound microscope with a ten ocular. 



The dishes when not under observation were kept in a moist 

 chamber consisting of a glass evaporating djsh with a cover. 

 This was kept in the tank with running sea water constantly 

 passing over it to maintain it at approximately the temperature 

 of the sea water itself. The water in each dish was changed 

 daily, actual tests showing th.ti a negligible changr in hydrogen 

 ion concentration occurred during this period. 



In general the cleavage stages of all separated blastomeres were 

 followed and sketched. After that the embryos were observed 

 at least once a day up to the fifth day. Several control eggs with 



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