838 THE NERVOUS SYSTEM. 



Tissues are first hardened from one to three weeks in Mliller's fluid, or simply in a 

 two or three per cent aqueous solution of potassium bichromate. They are then 

 transferred to a mixture of one-per-cent aqueous solution of osmic acid one part, 

 Milller's fluid two parts, where they remain from three days to a week. 



By following the lines of fat droplets, degenerative changes in nerve fibres may be 

 traced either in the peripheral nerves or in the central nervous system. 



Eofdn-Rffimatoxylin Staining. Suppurative inflammation of the central nervous 

 system and its membranes, or the connective-tissue changes in general, may be studied 

 in sections from the tissues hardened in Milller's fluid and alcohol, stained double with 

 haematoxylin and eosin (see page 59), and mounted in Canada balsam. 



Weigert's Method. A very useful method of staining sections of nerve tissue, espe- 

 cially of the brain and cord, is that known as Weigert's hcematoxylin method. The tissue 

 Is first well hardened in Milller's fluid. 



Blocks of the hardened tissue are embedded in celloidin and sections made in the 

 usual way. The sections are first soaked for twenty-four hours in a saturated aqueous 

 solution of neutral cupric acetate diluted with an equal bulk of water, or, if the material 

 has been kept some time and takes the haematoxylin stain with difficulty, a better result 

 is often obtained by soaking the sections for from twelve to twenty-four hours in a three 

 to five per cent aqueous solution of bichromate of copper before staining. They are 

 now thoroughly washed twice in water, then in alcohol, and then are transferred to the 

 haematoxylin solution, made as follows: 



Haematoxylin crystals 1 gm. 



Alcohol, 97 per cent 10 c.c. 



Water 90 " 



Saturated aqueous solution lithium carbonate 1 " 



In this solution the sections remain for two hours. (If the finer fibres of the cere- 

 bral cortex are to be brought out the sections must remain for twenty-four hours in the 

 haematoxylin solution.) The sections are now thoroughly washed in two or three 

 waters and transferred to the bleaching solution, composed as follows : 



Potassium ferricyanide 2.5 gm. 



Sodium biborate 2. " 



Water 200 c.c. 



In this fluid the sections discharge a brownish color, and they remain in it until the 

 gray matter has a distinct yellow color and the white matter is bluish-black. The 

 time required to produce this effect varies considerably, and is usually from half an 

 hour to an hour. The sections are now washed, dehydrated with alcohol, cleared up in 

 oil of cloves or oil of origanum, and mounted in balsam. The sections may be stained in 

 alum carmine before dehydration, to bring out the nuclei. In sections stained by this 

 method the gray matter, connective-tissue elements, and ganglion cells have a yellow 

 or yellowish-brown color, the axis cylinders are uncolored or have slight yellowish tint, 

 while the medullary sheaths are bluish-black or black. 



Nissl's Staining Method. There are several variations of this method, but the fol- 

 lowing gives good results in most cases : 



The essential feature of the so-called Nissl's method is the application of the aniline 

 dyes to the staining of certain structural elements in the nucleus and cytoplasm, which 

 are distinguished from the other structures of the cell by a differentiating decoloriza- 

 tion with alcohol. 



Methylene blue is the most generally useful of the aniline dyes for this purpose. 

 The specimens should have been carefully hardened in sublimate solution or in 

 alcohol or in formalin. 



Very thin sections are stained in one-per-cent solution of methylene blue. The 

 staining may be effected on a slide on which the sections are floating in the blue solu- 

 tion by gently heating over a lamp until the fluid steams. 



The sections are now transferred to a mixture of absolute alcohol 90 parts, 

 with aniline oil 10 parts, in which the differentiation is effected by the use of successive 

 fresh portions of fluid until slight but distinct differentiation in color is seen between 



