STAINS, ETC. 



1393 



STAINING OF NERVE-TISSUE 



formic glycerin. Freud's Method. Wash sections 

 of tissue hardened in Erlicki's fluid with water, and 

 place them for from 3 to 5 hours in a I per cent, 

 gold-chlorid solution. Wash with water and treat for 3 

 minutes with a solution of caustic soda, I part, in water, 

 5 or 6 parts. Drain, but do not wash, and place in a 

 10 per cent, solution of potassium iodid. Remove in 

 irom 5 to 15 minutes, wash in water, dehydrate, and 

 nount. Impregnation of axis-cylinders is obtained by 

 his process. Gerlach's Method. Harden pieces of 

 , phial cord for from 15 to 20 days in I or 2 per cent. 

 .mmonium bichromate. Make thin sections, and im- 

 nerse them in a solution of potassio-gold chlorid I part, 

 rater 10,000 parts, slightly acidulated with hydrochloric 

 del. In from 10 to 12 hours, wash in hydrochloric acid, 

 : 3000, and bring into a mixture of hydrochloric acid I 

 >art, and 60 per cent, alcohol 1000 parts, then for a few 

 ainutes into absolute alcohol. Clear and mount. Golgi's 

 jold Method. To demonstrate motor nerve- endings. 

 Treat the tissue for 1 or 2 minutes in a o. 5 per cent, solu- 

 ion of arsenic acid, and then for from 15 to 20 minutes 

 vilh ao.5 per cent, solution of potassio-gold chlorid, and 

 educe in the sunlight in a I per cent, solution of arsenic 

 citl. 1. Flechsig s Modification. Harden in a 2 per 

 ent. aqueous solution of potassium chromate, and im- 

 >regnate with a 1 per cent, solution of corrosive sublimate, 

 veeks or months, according to the size of the specimen. 

 \ 'lace the sections in 96 per cent, alcohol. Stain for from 

 to 8 days at 35 C. in the following : pure extract of 

 apanese redwood I gm., absolute alcohol IO c.c, dis- 

 Jled water 900 c.c, and 5 c.c. each of saturated 

 i olutions of tartaric acid and sodium sulphate. Each 

 lection is then placed in 3 c.c. of a o. 2 per cent, solution 

 If potassium permanganate until the purple color of the 

 tuid has faded out ; then decolorize in Pal's solution. 

 IVhen the yellow color has vanished, carry into a mix- 

 ure of a 1 per cent, potassio-gold chlorid solution 5 

 rops, and absolute alcohol 20 c.c. After the precipi- 

 ce of sublimate has turned black and the red tissue has 

 ecome blue, wash quickly in distilled water 20 c.c. and 

 5 per cent, solution of potassium cyanid I drop. De- 

 ydrate in absolute alcohol, and clear in lavender-oil. 

 he nerve-fibers are stained red, the ganglion-cells, with 

 leir processes, black. 2. Kit h ne' s Modification. Used 

 >r nerve-endings. Instead of using Golgi's gold solu- 

 on, place the tissue in the following : I per cent. 

 Dtassio-gold chlorid 12 c.c, 2 per cent, osmic acid 3 

 c. , 5 per cent, arsenic acid 60 c.c. From this bring 

 into I per cent, arsenic acid, and reduce in sunlight. 

 he tissue may be preserved in May's fluid. (See Ex- 

 ttination and Pi-eservation Media). Golgi's Silver 

 lethods. 1. Soak pieces of perfectly fresh spinal 

 )rd in a 2 per cent, potassium bichromate solution, for 

 om 8 to 15 days in summer and for about one month 

 winter. Wash them, and put them into a 0.75 per 

 nt. solution of silver nitrate ; in warm weather the 

 action will be complete in 2 or 3 days, in from 8 to IO 

 ivs in winter. Dehydrate in alcohol, section if ne- 

 ssary, clear in oil of turpentine, tease in turpentine, 

 id mount in dammar. The preparations are then ex- 

 tsed to diffused daylight or to direct sunlight, to effect 

 condary impregnation. By this method may be de- 

 onstrated the chain of conical funnels, set one within 

 , e other, and embracing the axis-cylinder with their 

 irrow apertures. Somewhat greater precision of re- 

 gion is obtained by interstitial injection of the fresh 

 'Sue with osmic acid before placing in the bichromate 

 lution. 2. For the study of peripheral nerves, the 

 ^cess is modified as follows : Immerse pieces of nerve 

 the bichromate solution for from 4 hours to 2 days ; 

 insfer them to the silver bath, in which they should 

 main for from 12 to 24 hours. Wash with successive 



alcohols, tease in alcohol, dehydrate, clear with tur- 

 pentine, and mount in dammar. Reduce in direct sun- 

 light. The preparations are permanent, but the results 

 are not so fine as in the following method. 3. Place 

 a piece of fresh nerve in a mixture of 10 parts of a 2 

 per cent, solution of potassium bichromate and 2 parts 

 of a 1 per cent, solution of osmic acid ; after an hour's 

 immersion, cut into lengths of from ]/ 2 to 1 cm., and 

 return to the solution. Four hours after the first immer- 

 sion, begin to add pieces of silver nitrate to the bath, 

 and, from time to time, transfer pieces of nerve, so as 

 to ensure the proper duration of immersion for some of 

 the pieces. The duration of the silver bath should not 

 be less than 8 hours, and may be indefinitely prolonged. 

 The strength of the silver solution should be 0.5 per 

 cent. 1. Obregia 1 s Modification. The sections are 

 transferred from absolute alcohol (after they have been 

 in sublimate or silver solution) directly into 10 c.c. of 

 absolute alcohol containing 8 or 10 drops of a 1 pier 

 cent, solution of gold chlorid. The latter should be 

 made half an hour before and exposed to diffuse light. 

 The specimens in the solution are kept in the dark for 

 from 15 to 30 minutes, then washed rapidly in 25 per 

 cent, alcohol, then in water, and for 5 or 10 minutes (not 

 longer) in 10 per cent, solution of sodium sulphid. They 

 are again washed in water, and stained with carmin, 

 hematoxylin, or Weigert's stain, and mounted in balsam. 

 2. Ramon y CajaP s Modification. Small pieces of 

 brain are fixed for from 12 to 24 hours, in the dark, in 

 potassium bichromate 3 parts, osmic acid, I per cent., 

 25 parts, water 100 parts. An abundant quantity of 

 the fluid should be used, and changed several times 

 during the first day. Embiyonic tissue requires from 

 12 to 24 hours' immersion, adult tissue from 2 to 3 

 days. After hardening, which must not be excessive, 

 wash in a 0.25 per cent, solution of silver nitrate, for 

 15 minutes, and then place in a 0.75 per cent, solution 

 of silver nitrate containing I drop of formic acid to each 

 100 c.c. (/<*« Gehachten). The tissue may be left in 

 the silver bath for from 36 to 48 hours. The silver 

 will be thrown down as a very fine precipitate of silver 

 bichromate. 3. Sa/a's Modification. Place the tissue 

 for 4 or 5 days in a 2 per cent, solution of potassium 

 bichromate, for from 24 to 30 hours in 8 parts of the 

 bichromate solution and 2 of the osmium solution, then 

 in a silver bath of 0.75 per cent, strength. Wash with 

 water, fix to a cork with gum, harden in alcohol for a 

 few hours, and cut without embedding. 4. Se/irzta/d's 

 Modification. Prior to bringing the tissues into the 

 silver bath, put them into a 10 per cent, solution of 

 gelatin in water; they may be embedded in the gelatin 

 in a paper tray, with the aid of a little heat, and thus 

 brought into the silver solution. The gelatin is re- 

 moved by warm water saturated with silver chromate. 

 This process prevents the formation of the precipitate 

 that frequently occurs at the margins of the preparation 

 in Golgi's method. Samassa holds that the precipita- 

 tion may be prevented by preserving the preparation 

 without a cover. Fick and Huber recommend that the 

 use of an aqueous fluid be avoided and that the section 

 be mounted without a cover, or that the cover be raised 

 from contact with the slide by means of wax feet, or 

 that the balsam be rendered anhydrous (by heating it 

 on the slide with the specimen) and the cover put on 

 in the usual way. 5. Van Gekuchteh 's Modification. 

 This process is like that of Ramon y Cajal, with this 

 difference : I c.c. of formic acid is added to each 100 

 c.c. of the silver bath, to assist its action. Twenty- 

 four hours' immersion is sufficient. The tissue is then 

 washed, treated for 15 minutes with alcohol, then for 

 the same time with absolute alcohol, and for the 

 same time with celloidin solution, after which it is 



