4 o6 APPENDIX. 



The Weigert method requires the employment of three solutions, the 

 mordant (A), the stain (B), and the differentiation fluid (C), made respec- 

 tively as follows: 



A. Saturated aqueous solution of neutral cupric acetate 



approximately 10 gm. of the copper salt to 100 cc. dis- 

 tilled water. 



B. Hematoxylin crystals i gm. 



Absolute alcohol 10 cc. 



Distilled water 100 cc. 



The hematoxylin is dissolved in the alcohol, added to the dis- 

 tilled water and boiled ; after cooling; filter. 



C. Borax 2 gm. 



Potassium ferric cyanide 2.5 gm. 



Distilled water 100 cc. 



After being cut in celloidin, the sections are placed for 12 hours in the 

 copper- solution, composed of equal parts of freshly filtered A and distilled 

 water. They are then transferred directly to the stain, B, in which they re- 

 main for 12-36 hours, followed by differentiation in C. The over-stained 

 sections remain in this fluid until color is no longer extracted and the con- 

 trast between the white and gray matter is well accentuated, ordinarily from 

 30-60 minutes sufficing. The sections are then thoroughly washed in run- 

 ning water for 812 hours and carried through ascending alcohols until de- 

 hydrated, when they are cleared in carbol-xylol and mounted in balsam. If 

 the staining has been unsuccessful, the same sections may be placed in Miil- 

 ler's fluid for 24 hours, rinsed in distilled water, and treated with the copper 

 and subsequent solutions as before. 



Sodium Carminate. Satisfactory as is hematoxylin-eosin for usual 

 purposes, this stain is inadequate for really good demonstrations of the nerve- 

 cells and nerve-fibres of the brain and spinal cord. Excellent preparations 

 of these elements may be made by the following method : Small pieces of 

 the fresh tissue, not over i cm. thick, are fixed in an excess of M tiller's fluid, 

 changed daily during the first week and occasionally afterwards. After four 

 weeks the tissue is transferred directly, without washing, into a sufficient 

 quantity (40-50 cc. ) of i p.c. aqueous solution of sodium carminate, in 

 which it remains 3 days with frequent shakings. The pieces of stained tissue 

 are washed for 24 hours in running water, and then dehydrated in ascending 

 alcohols, embedded in celloidin and cut. When successful, the cells and the 

 axis-cylinders appear red and sharply differentiated on a light ground, the 

 cells of Purkinje in such preparations being beautifully shown. Lack of pene- 

 tration and over-staining at the surface are the most common sources of 

 failure. 



Chrome-Silver Impregnation. The introduction by Golgi of 

 methods of silver impregnation has added an important means of demon- 

 strating the astonishing richness and extent of the ramifications of the neu- 

 rones and of the neuroglia-cells. Of the several procedures suggested by 

 Golgi the so-called rapid method is here given. It is most successful when 

 applied to the nervous tissues of the foetal or new-born animal, and, at best, 

 is capricious and uncertain. 



The fresh young tissue is fixed either in bichromate-formalin (4 parts of 

 3.5 p.c. aqueous solution of potassium bichromate to i of 40 p.c. formalin), 

 or in Golgi s fluid (9 parts of 3.5 p.c. aqueous solution of potassium bichro- 

 mate to i part of 2 p.c. aqueous solution of osmic acid). 



