THE ACTION OF MORDANTS 117 



Similar linkages are likely to be formed with carboxyl and other 

 acid groups in proteins and other tissue-constituents. 



It is not always necessary to prepare a mordant/dye complex 

 first and subsequently to allow it to come into contact with the 

 tissues. On the contrary, one may first soak the tissues in a solu- 

 tion of the mordant and then in a solution of the dye. The basiphil 

 tissue-constituents will take up the positively charged hydrated 

 aluminium ion and the dye will displace water from the attached 

 aluminium complex when given the chance to do so. It might, 

 indeed, be said that the dye is being used as a reagent for the 

 detection of aluminium (though one might be misled if the tissues 

 happened to contain another metal capable of mordanting the 

 dye). Both the single-bath method (in which the dye is mixed with 

 the mordant) and the two-bath method (in which the tissue is put 

 first in the mordant and then in the dye) are of common use in 

 microtechnique. 



When the single-bath method is used, one may dye either 

 regressively or progressively, but regressive dyeing is almost in- 

 variable with the two-bath process. There are two chief ways of 

 differentiating mordant dyes after deliberate over-dyeing. One 

 may extract the excess of dye by placing the tissue either in an 

 acid (often a weak solution of hydrochloric acid) or else in a solu- 

 tion of the substance that was used to mordant. 



Acids attack both the links in the tissue /mordant /dye complex. 

 It is easy to prove that they attack the tissue /mordant link.^* 

 Haematein (p. 94) is a convenient dye for experiments of this 

 kind. It is only necessary to take two slides, treat both of them 

 with the mordant, then one only with an acid, and finally, after 

 washing away the acid, to put both slides in the dye for the same 

 length of time. The section that has been in the acid will be 

 more feebly dyed than the other, because the acid has removed 

 part of the aluminium from the tissues (and would remove it all, 

 if given sufficient time). The hydrogen ions of the acid compete 

 with the positively charged water /aluminium complex for attach- 

 ment to the negatively charged sites in the tissue. 



That acids also attack the mordant/dye link is very easily shown 

 with those dyes that change their colour when they associate 



