THE DEVELOPMENT OF THE VENTRAL NERVES IN SELACHn. 297 



the embryos violently, for they become extremely brittle and fragile. All subsequent 

 changes, including the embedding, should, therefore, be made in a single glass dish. 



2. The fixing fluid is siphoned off and replaced by methyl alcohol for five minutes. 

 According to vom Rath this bath may be safely omitted. 



3. The methyl alcohol is replaced by a weak (about .5%) solution of pyrogallic, 

 acid or by crude pyroligneous acid in which the embryos are kept for six to twenty- 

 four hours in the dark. Preparations made with the pyroligneous acid, the action of 

 which is somewhat uncertain and difficult to control, are apt to be overblackened, 

 but are more permanent than those made with pyrogallic acid. The best preparations 

 of large (over 30 millimetres) embryos were obtained by the use of pyroligneous acid 

 in which they remained twenty-four hours after a four days' immersion in vom Rath's 

 fluid (A). 



4. The embryos are subsequently passed slowly through the alcohol grades 

 35%, 50%, 70%, 85%, and 95%, in which they remain with occasional change to 

 fresh fluid until the picric acid ceases to be extracted, a process that takes weeks. 



5. The embedding should be done in very hard paraffin, as it is necessary to cut 

 thin sections, and the embryos are so brittle that they break badly when cut in the 

 usual grades. 



While there is considerable variation in the action of the method upon different 

 embryos, successful preparations show the nervous system to be of a steel-gray color, 

 and the muscles slightly yellow from the picric acid. Sections should be mounted 

 in balsam with or without a cover-glass. When a cover-glass is used the sections 

 near the edge of the cover-glass retain their stain perfectly, while those near the centre 

 of the cover-glass fade in time to a dull yellow. 



Hermann's fluid used instead of vom Rath's mixture gives very similar results, 

 but the shrinkage of the embryos is much greater. The shrinkage is less with 

 Flemming's fluid, but the latter does not permit of subsequent treatment with pyro- 

 gallic acid. Treated by either of these two methods without pyroligneous or pyro- 

 gallic acid, the embryos deteriorate somewhat when kept in paraffin, so that they 

 stain much less readily with safranin than when cut soon after embedding. Material 

 treated by the vom Rath method and preserved in paraffin showed no signs of fading 

 or deterioration after five years. 



The vom Rath method has the following advantages and disadvantages: First, 

 it causes less shrinkage or swelling than any embryological method known to the 

 writer. Neuraxones are stained deeply in the early stages of development and cell 

 boundaries are brought out with beautiful distinctness. On the other hand, the 

 tissues are so blackened that very thin sections are necessary. The method is in- 



