MYELIN STAINS 529 



imbedding). The sections are treated with ammonia (20 

 drops to 50 c.c. of water, which clears up the general mass of 

 the brain substance, leaving the medullated fibres black. The 

 preparations are not permanent, unless (Ranvier, Traite, 1 ed., 

 p. 1086) they are fixed for a quarter of an hour in osmic acid 

 vapour. 



1071. Greenfield and Carmichael {Brain, Iviii, 1935, p. 483). 

 found this method the most satisfactory for the finest myelin 

 sheaths in small peripheral nerves. These do not need to be left 

 in the osmic acid for more than twenty-four to forty-eight hours 

 and after sectioning in paraffin or celloidin are mounted from 

 95 per cent, alcohol in Gurr's neutral-mounting medium. 



Azoulay's Osmic Tannic Acid Methods (Anat. Anz., x, 1894, p. 25). 

 (A) Sections of old Miiller material are put for five to fifteen minutes 

 into a solution of 1 : 500 or 1 : 1000 of osniic acid, rinsed with water, 

 and put for two to five minutes into a 5 or 10 per cent, solution of 

 tannin, warming them therein over a flame till vapour arises, or in 

 a stove at 50° to 55° C. Wash for five minutes in water, eounterstain 

 with carmine or eosin, and mount in balsam. If the sections are too 

 thick it will be necessary to differentiate by Pal's process, or with eau 

 de Javelle diluted with 50 volumes of water. (B) Material that has been 

 in an osmic mixture (fluids of Flemming, or Marchi, or Golgi). Sections 

 as before, then tannin bath, warming for three to ten minutes, the rest 

 as before. 



Hellier and Gujmpertz (Ztschr. wiss. Mikr., xii, 1895, p. 385) give 

 for peripheral nerves, and Heller (ibid., xv, 1898, p. 495) for central 

 nervous system, the following method. Sections of Miiller material 

 are put into 1 per cent, osmic acid (twenty-four hours at 37° C. for 

 peripheral nerves ; ten to thirty minutes, at room temperature, for 

 central nerve-fibres). They are treated with pyrogallie acid (a photo- 

 graphic developer will do) till the nerve-fibres are black, then with a 

 violet-coloured solution of potassium permanganate till the sections 

 become brown, then with 2 per cent, oxalic acid till they become yellow- 

 green. Wash out well between each operation. 



Robertson {Brit. Med. Journ., 1897, i, p. 651) hardens tissues in 

 Weigert's gliabeize for ten days or longer, and cuts sections either by 

 freezing or after imbedding in celloidin. He places these in 1 per cent, 

 osmie acid in the dark for half an hour, then in 5 per cent, pyrogallie 

 acid for half an hour, and differentiates by Pal's method. (This method 

 was used exclusively for the sections reproduced in Bruee's " Atlas of 

 the Spinal Cord.") 



Orr {Journ. Path, and Bad., vi, 1900, p. 387) stained the fine cortical 

 fibres by fixing very thin slices of cortex for forty-eight hours in 2 per 

 cent, osmie acid, 8 c.c. 1 per cent, acetic acid, 2 e.e., reducing the osmic 

 acid in 10 per cent, formol, and imbedding in celloidin or paraffin. 



FiNOTTi {Virchovo's Arch., cxliii, 1896, p. 133) makes sections of 

 material that has been in Miiller's fluid for not more than a few weeks 

 or months, and puts them for four to ten hours (in the dark) into a 

 freshly-prepared mixture of 1 or 2 parts of 1 per cent, osmic acid, and 

 1 part of a concentrated solution of picric acid in one-third alcohol. For 

 peripheral nerves ; myelin (normal), black. 



WiTTiMAACK {Arch. Ohrenheilk, Ixi, 1904, p. 18) mordants till green 

 (temporal bones) in 90 parts of Miiller's fluid with 10 of formol and 3 

 to 5 of acetic acid, decalcifies with nitric acid and formol, treats sections 



