132 STAINING. 



the staining agent. It will readily be understood that the pro- 

 duction of an insoluble dye salt renders solution in water more diffi- 

 cult, but this alone is not enough. The compound must also be 

 firmly attached to the surface. Thus, the barium salts of colour 

 acids are insoluble, but barium salts cannot be used as mordants for 

 acidic dyes ; the compound formed is readily washed off mechani- 

 cally. 



The insoluble compounds between a dye and a mordant are known 

 as " lakes." But the chemical nature of these substances is by no 

 means clear especially when they are produced in situ in stained 

 tissues. In fact, they do not behave as simple compounds of the 

 dye and the mordant. They resist the action of strong acids and 

 bases in moderately strong solutions. So that the stains obtained 

 by this method are characterised by durability and " fastness." 



On the whole, we must conclude that there are factors still un- 

 known, but probably related to the electrical properties of the 

 surface, which play an important part in these reactions with 

 mordants. A simple chemical theory does not account for all the 

 phenomena. Changes in the properties of the surface by the action 

 of the mordant are concerned. In this connection, we may call to 

 mind how frequently multivalent cations are used as mordants. 

 Alum is one of the commonest of these. 



A further mention may here be made of the " progressive " and 

 " regressive " methods, between which there is a more definite 

 distinction than that between " substantive " and " adjective " 

 staining. A preparation may be placed in a very dilute solution of 

 a dye and the action stopped when the elements with the greatest 

 " affinity " for the dye have taken it up. The staining of nuclei 

 with dilute hsematoxylin-alum may be mentioned. If the action is 

 prolonged, various other constituents of the cell, besides the nucleus, 

 take on the colour. As a rule, no great differentiation is obtained 

 by this method. Better results are given by the " regressive " 

 method, in which a general overstaining is followed by a partial 

 decoloration, in which certain elements retain the stain, owing to 

 special chemical or physical properties, after it has been removed 

 from the rest. Thus safranin stains the whole section of a deep red 

 colour ; acting on it with alcohol removes the stain from all but the 

 chiomatin and the nucleoli. This action of alcohol may be explained, 

 as already pointed out, by its effect on the magnitude of the electric 

 charge, since its dielectric constant is lower than that of water. 

 Other differentiating agents are also used. Iron-alum, in the iron- 

 hsematoxylin method, serves both as preliminary mordant and as 



