36 



always in the form of a separate solution (e. g., aluminium hema- 

 toxylin) and in many cases the mordantage is given in the fixing. 

 Mordantage, directly or indirectly employed, is also useful in staining 

 by the progressive method. Stains or dyes that do not require a 

 mordant are termed Substantive, those requiring a mordant to make 

 them "take" are Adjective stains. 



Delicate results in exact differential staining by either the progres- 

 sive or regressive methods can best be secured only by section stain- 

 ing, although differentiation of the sections after in toto staining may 

 be resorted to. 



82. Differentiation. In the regressive method it is neces- 

 sary to remove the excess of stain by the application of a solution 

 that will usually differentiate it, or bring out the selective action of 

 the stain. A small amount of differentiation is usually necessary 

 in any case for the most delicate results. Alcohol, 95% or other 

 grade, may often be used as a differentiator. 95% acidified with 

 hydrochloric acid (1/10 to 1%) is sometimes used, or^a special dif- 

 ferentiator is required (acetone, alum solution, clove-oil, etc.). 

 It is usually necessary to control the differentiation by use of the 

 microscope. 



83. Impregnations. In addition to the typical methods of 

 coloring tissue by means of stains there is a group of methods in 

 which the coloring matter is deposited in the cell or tissue that it is 

 desired to differentiate, in the form of a precipitate. These are 

 known as impregnation methods and are of great value, especially 

 as applied to nervous tissues. A hard and fast line, however, can- 

 not be drawn between true staining methods and impregnations. 

 Silver nitrate and gold chlorid are the substances most generally 

 employed in the impregnation of tissue ( 221, 223). 



84. Choice of stains. Remember that the staining is greatly 

 affected by the previous treatment, brilliancy or selectivity in the 

 result being in many cases dependent on the fixer employed or im- 

 paired by the improper or incomplete washing out of the fixer. 



In staining, therefore, consider three things, (a) what it is de- 

 sired to bring out, what kinds of stains you need to employ, (b) 

 the mode of fixation that has been emploj^ed, and (c) the imbedding 

 method must also be considered in the choice, since if celloidin is 

 employed certain stains that color it deeply should be avoided. 



