542 



METHODS AND FORMULAS 



MS 30 



niide as may have been present was 

 then reduced to the metalHc form by a 

 photograpliic developer. It is doubtful, 

 however, whether or not metallic silver 

 is the end product of any of the reac- 

 tions employed in modern silver-staining 

 techniques. 



There is no question in photography of 

 any intermediate condition between the 

 bromide and the metal, the various grada- 

 tions of shade being dependent on the 

 total mass of silver present. Many of the 

 silver-staining techniques, on the con- 

 trary, result in varying shades of brown, 

 making it more probable that the end 

 product is some dark colored silver pro- 

 teinate, a hypothesis born out by the fact 

 that some of these stains may be differ- 

 entiated by exposure to distilled water. 

 Thus, in the techniques of del Rio-Hor- 

 tega (MS 33.31 del Rio-Hortega 1925) 

 mitochondria are demonstrated by the 

 process of washing out all impregnated 

 material, other than mitochondria, in 

 water. 



In the absence of any accurate informa- 

 tion as to the method by which the results 

 are produced, it is best to divide the tech- 

 niques into four great classes according to 

 the reagents employed. In the first class, 

 silver is applied to the tissues as a solu- 

 tion of silver nitrate to which, in some 

 modifications, may be added alcohol or 

 pyridine. Moreover, in many cases, so 

 little pyridine is added that the amount 

 of silver-pyridinium complex present is 

 insignificant. The second class comprises 

 the Bodian techniques in which silver is 

 employed as the proteinate. The third 

 class contains those techniques in which 

 the application of silver nitrate is either 

 followed, or replaced, by immersion in so- 

 lutions containing silver-diammine com- 

 plexes, secured usually by dissolving either 

 silver hydroxide or silver carbonate in 

 ammonia. The fourth class involves prior 

 treatment of the tissues with a solution 

 containing some other metalhc ion, such 

 as a chromate or dichromate, with which 

 the silver subsequently applied is known 

 to react. 



The first class, designated below as MS 

 31, is usually referred to as the "Cajal 

 technique," although this name is also 



associated with the third class. Cajal 1907 

 (21344, 8:21) distinguished eleven meth- 

 ods by which his results could be pro- 

 duced, and divided the possible reactions 

 into eight classes. In broad outUne it may 

 be said that these techniques involve 

 either the exposure of fresh tissues to silver 

 nitrate, and the subsequent reduction of 

 the absorbed silver to a dark-colored com- 

 plex by exposure to formaldehyde, or al- 

 ternatively, the prior treatment of the tis- 

 sues with a series of "accelerators," the 

 purpose of which is to cause a greater 

 differentiation of types of nervous struc- 

 tures. These techniques are used prin- 

 cipally for the demonstration of nervous 

 structures ; the various modifications which 

 have been proposed have been designated 

 to bring one type of structure more into 

 prominence than another. Much of the 

 classic work in tracing nerve tracts, and 

 in demonstrating fine nerve endings in 

 tissue, was carried out by these methods. 

 This, as are all the other classes, is sub- 

 divided according to the purpose for 

 which the technique is intended. 



The second class of stains employs a 

 relatively new method, in which silver is 

 adsorbed on the tissues from a silver pro- 

 teinate solution, sometimes in the pres- 

 ence of metallic copper. These techniques 

 have the advantage that no special prepa- 

 ration of the tissues is required and they 

 may therefore be used on ordinary paraffin 

 sections. 



The next great class, designated MS 33 

 below, is of the most varied application. 

 Workers in this group, with which the 

 names of Bielschowsky, Cajal, and del 

 Rio-Hortega are associated, were able to 

 adapt silver-staining techniques to the 

 demonstration of the connective tissues 

 of the central nervous system, through 

 the discovery that prior treatment of the 

 tissues with a variety of solutions (vary- 

 ing from uranium nitrate to alcoholic ex- 

 tract of cork crumbs) prevented the stain- 

 ing of nervous elements and brought into 

 contrast their supporting structures. If the 

 silver is dei)osited from a colloidal en- 

 vironment, which may be produced either 

 from solutions of gelatin or in other ways, 

 there is a complete inhibition of the ab- 

 sorption of silver, either by nerves or 



