No. I.] STUDIES ON LIMULUS. 1 03 



2. Methods. 



The results obtained in our work upon Limulus have been 

 obtained largely by careful dissection. In order to accom- 

 plish much by this method, however, it has been necessary to 

 prepare the material in a special way. In fresh specimens the 

 nerves were found to be so transparent, and the other tissues 

 so tough, that it was impossible to trace out the smaller nerves 

 Myth any degree of accuracy, and in most alcoholic material the 

 clotted blood and organic precipitates upon the tissues rendered 

 it difficult to distinguish the smaller nerves from arteries. 



Specimens which had been for a long time (two or three 

 years) in alcohol of from 50 per cent to 70 per cent were found 

 to be in remarkably good condition for dissection. The nerves 

 were white, and easily traced in the partially macerated tissues 

 when dissection was carried on under water or weak alcohol. 



Equally good material was procured by taking large female 

 Limuli at the end of the spawning season, when all the 

 ova had been shed, and treating them in the following man- 

 ner. They were first allowed to bleed freely, then cut in halves 

 along the median line, and the parts macerated for several 

 days in water. Finally, they were transferred to 70 per cent 

 alcohol until ready for use. In this way many of the organic 

 substances, which would have been precipitated by the alcohol, 

 were dissolved out. The alcohol whitened the nerves and made 

 them stand out in contrast with the other tissues. , It was diffi- 

 cult to determine the proper length of time to continue the 

 maceration, as it varied with different specimens and with the 

 temperature. A number were tried and the best selected. 



Dissection was carried on under water or weak alcohol with 

 the aid of a lens, fine pointed forceps, and needles. When- 

 ever any doubt arose in regard to the character of the tissues, 

 the doubtful portions were excised, stained, and examined under 

 a compound microscope. 



Injected specimens were used in tracing out the arteries. 

 A great deal of the work of dissection was verified by exami- 

 nation of Dr. Patten's serial sections, both longitudinal and 

 transverse, of young crabs, from i to il- inches long exclusive 



