PRENTISS: THE OTOCYST OF DECAPOD CRUSTACEA. 179 



the fixative alone for three to five days, and washing out for at least two 

 weeks iu 90 % alcohol. The myelin sheath was intensely blackened, 

 while all other tissues remained a yellowish brown. 



For tracing nerve fibres, both to peripheral and central endings, intra 

 vitam staining proved of most value. Different methods were employed 

 for obtaining peripheral and central stains. A one per cent solution of 

 methylen blue in normal NaCl was injected into the body in either case. 



For peripheral endings several injections were made into the abdomi- 

 nal blood space, at intervals of thirty minutes. When the animals 

 showed signs of stupefaction, a final injection was introduced into 

 the pericardial chamber. The amount of solution injected varied 

 from a few drops, iu Palfemonetes, to five cubic centimetres, 

 in the lobster. In from 15 to 30 minutes after the final injection. 

 the animals were usually dead. The part to be studied was then dis- 

 sected out, barely covered with normal salt solution, and examined from 

 time to time under the microscope, until a satisfactory degree of stain- 

 ing had been reached. 



For central terminations one injection only was made, and this 

 directly into the chamber of the heart, only a few drops of the solu- 

 tion being required. When the blue color was well difi'used throughout 

 the tissues (about one hour after injection), the brain was dissected out, 

 or exposed, and examined as before. For fixation of the stain Bethe's 

 ammonium-molybdate method for invertebrates was used. It was found 

 to be better to leave preparations in xylol for only the shortest possible 

 time, as this reagent diffuses the color. Preparations fixed by this 

 method keep very well for a year or more, but after this they ultimately 

 deteriorate, fibres originally sharp and continuous in outline becoming 

 mere dotted lines, while the surrounding tissues take on a deep yellow 

 hue. When both brain and otocyst were examined together, the peri- 

 pheral cells and fibres stained first, then central fibres, central termina- 

 tions, and ganglion cells of the brain in the order named. Sections 

 60-120 |U in thickness were cut, but by far the greater number of pre- 

 parations were examined in toto. The transparency of the tissues made 

 this possible even with the brains, a millimetre or more in thickness, of 

 large crayfish. 



To get constantly complete impregnations of both peripheral and cen- 

 tral endings, it is necessary to expose to the atmosphere the part to 

 be studied. The impregnation then takes place sooner, lasts longer, 

 and affects a larger number of elements. The fixation of the color is 

 also much better in this case, because the fluid can penetrate much more 



