154 PAUL S. McKIBBEN 



The method used is with some modification that given by- 

 Wilson ('10). The animals are tied by the legs to a board, the 

 root of the tail being held immovable by a wooden clamp. The 

 tail is cut off and after bleeding the stain is injected through the 

 caudal artery. A 0.066 to 0.075 per cent solution of methylene 

 blue (Griibler's ''med. pur." or "rect., nach Ehrlich") in salt 

 solution was found most useful. 



Methylene blue (0.5 per cent in water) 13-15 cc. 



Salt solution (0.75 per cent in water) 87-85 cc. 



About 200 CC. of the stain is usually injected into an animal 

 of 40 cm., outlet being furnished by the caudal vein and by 

 cutting the tips of the gills to which clamps are applied when 

 the vessels are well filled. Pressure is obtained by gravity and 

 for a good injection of vessels of the head a pressure is necessary 

 which is sometimes sufficient to rupture abdominal capillaries. 

 After the vessels are well filled and the outlets cut off, the animal 

 remains untouched for three to five minutes. The head is then 

 cut off, the lower jaw removed and the region to be studied 

 moistened with salt solution and exposed to the air. After a 

 successful injection color appears in the eye-muscle nerves almost 

 immediately on exposure to the air. The dye in the other nerves 

 is oxidised somewhat later. 



For fixation, if permanent preparations are desired, a cold solu- 

 tion of ammonium molybdate (8 per cent in water) is used. This 

 is applied in an ice box for eighteen to forty-eight hours. (Some 

 material, thus fixed, has been kept successfully, for purposes of 

 dissection, in 4 per cent neutral formaldehyde — 9 parts water, 

 1 part, 40 'per cent formaldehyde — for four or five days without 

 total loss of the stain in the nerves). The molybdate is now 

 removed by washing in cold running tap water or by numerous 

 changes of cold water in the ice box and the tissue then trans- 

 ferred to several changes of 96 per cent alcohol in the ice box 

 and then to absolute alcohol. Clearing is accomplished in xylol 

 and the tissue mounted in balsam or embedded in paraffin. If 

 kept in the dark, preparations will keep for several years. For 

 further details concerning this method, see Wilson ('10). 



