No. I.] THE CRANIAL NERVES OF AMPHIBIA. 211 



APPENDIX ON TECHNIQUE. 



Although nearly every one who takes up any especial line of work 

 evolves, to a certain extent, his own technique, and although the Golgi 

 method is described in a number of articles and books, yet it may be well 

 to give the details of manipulation, as found convenient in my experience, 

 to benefit those who are not familiar with the subject and literature. 



1 . Hardening of Small Pieces in Osmium-bichrojnate. — The size of 

 the pieces will naturally depend upon the character of the tissue, but, as a 

 rule, one dimension should not exceed 3 to 4 mm. Perhaps the best mixture 

 for general use is potassium bichromate, 3>^%, 4 vols. + osmic acid, 1%, 

 I vol. The time of hardening must be largely a matter of experience, 

 depending upon such factors as the character of the tissue, its stage of 

 development, whether embryonic or adult, temperature, and the character 

 of the impregnation aimed at. In general, this time will lie between 2 and 

 5 days, embryonic tissue requiring less than adult. For the cortex of an 

 8-months' human embryo, however, I have found from 3 to 5 days — and 

 especially the latter period — gives the best results when using Berkley's 

 mixture. 30 to 50 cc. are required for a medium-sized tadpole. It is best 

 to put the specimen in a smaller quantity first, — a solution that has been 

 used once will answer, — and then change after an hour or so, putting the 

 specimen in the full quantity. The fluid should be changed at any time if 

 it becomes cloudy or ceases to smell quite strongly of the osmic acid. 



2. The Silver Bath. — 1% may be taken as a standard solution. The 

 pieces of tissue should be washed in several changes of the silver solution 

 (that which has been used once being available), until, after 10 to 15 

 minutes, the fluid ceases to cloud up with the silver chromate, which is 

 formed when the bichromate and silver solution meet each other. The 

 pieces should be left in a liberal supply of the silver solution — at least 

 double the quantity of osmium bichromate which has been used. Impreg- 

 nation takes place in 24 to 12 hours, or even less, but it is well to allow the 

 tissue to remain in the silver several days in the dark. Keeping the tissue 

 in the dark in i. and 2. is not really essential to obtain the reaction, but is 

 preferable, especially if it remains in the silver bath some time (see below). 



3. Cutting and Mounting. — {a) The pieces are transferred imme- 

 diately, and left Yz to i hour in 95% alcohol, this being changed several 

 times in the meanwhile; {b) next ^ to i hour in absolute alcohol; {c) 10 

 to 15 minutes in alcohol and ether, equal volumes; (^) ^ to i hour in 

 thin celloidin; (e) a few minutes in thick celloidin; (/") mounted on a 

 microtome block, and the celloidin hardened in chloroform. This process 

 applies especially to the tadpole, which, when put in the alcohol, is cut 

 transversely into several more pieces to facilitate the washing and, especially, 

 the penetration of the celloidin. For solid tissues, especially the central 

 nervous system, it is better simply to gum them to the block after the wash- 

 ing in alcohol, using celloidin and hardening it by only a short immersion in 



