Hardestv, spinal Nerves of the Frog. loi 



came necessary to return to osmic acid as offering the greatest 

 possibilities of obtaining satisfactory results. 



In order to obviate the difficulties attending its use, several 

 osmic acid mixtures were tried. These again had to be 

 abandoned as inefficient. Where one was an advantage in one 

 respect it was a drawback in another. Various mixtures of 

 osmic acid and formalin were tried, but, while giving cylindrical 

 and well blackened fibers, had to be abandoned because forma- 

 lin was found not only to accelerate the reduction of the osmium 

 but, what was not desired, also to produce a brittleness which 

 made it more difficult to obtain sections thin enough for photo- 

 graphing and reliable counting. 



The most promising combinations with osmic acid were 

 found in the application of a reducing agent after the exposure 

 to the action of the acid. After trying several of the reducing 

 reagents used in the ordinary photographic developers, the best 

 results were obtained with a o. 5 ^ water solution of pyrogallic 

 acid. If the nerve be washed for two and a half or three hours 

 in distilled water after removal from the osmic acid and then 

 placed for an hour or so in this solution of pyrogallic acid, a 

 decided increase is noticeable in the intensity with which the 

 medullary sheath is blackened, without any indication of a re- 

 duction of the osmium in the other structures of the nerve. 

 Some of the nerves used in making the counts of fibers recorded 

 in this paper had been treated in this way. 



The method of procedure which proved the most satisfac- 

 tory is the following. 



Dissection. 



The animal was killed and weighed and, if female, the 

 weight of the ovaries deducted. Then the viscera, the skin, 

 the limbs and a portion of the abdominal and thoracic muscles 

 were removed. This left only the head and the more dorsal 

 portion of the body. From the ventral side, the spinal cord 

 was now laid bare by carefully severing the pedicles of each 

 vertebra and thus removing the centra of the entire vertebral 

 column. This also lays open the intervertebral foramena. Then 



