220 DYEING 



been, its combination with a dye would have rendered micro- 

 scopical study impossible. 



Lakes can be used progressively. Hansen's iron haematein and 

 the oxazine lakes are ordinarily used in this way. It is, however, 

 almost invariable to work regressively when mordant and dye are 

 applied separately. 



There are three separate methods by which differentiation can 

 be carried out when mordant-dyes are used regressively. The 

 agents used are mordants, acids, and oxidizing agents. 



The tissue/mordant/dye complex is broken by excess of the 

 mordant. The dye distributes itself partly as a soluble lake with 

 the free mordant and partly as a component of the insoluble 

 complex; and since the amount of mordant in the complex is 

 small in comparison with the amount in the differentiating fluid, 

 nearly all of the dye will eventually associate itself with the latter, 

 if enough time be allowed. Since there will be much dye in some 

 tissue constituents (the chromosomes, for instance) and less in 

 others (such as the cytoplasm), a particular degree of differentia- 

 tion will leave no visible dye in certain parts though others remain 

 strongly coloured. 



Since a mordant will continue to extract dye even when it has 

 already taken up a certain amount of it, but will colour tissues 

 powerfully if it is heavily laden with dye, there must be an inter- 

 mediate amount of dye that will cause strong colouring of basiphil 

 objects but not of those that have less affinity for the lake. This ex- 

 plains the facility with which certain lakes can be used. Grenacher's 

 alum carmine ^^^ and Mayer's carmalum ^^^ may be quoted as 

 familiar examples. Chromatin is coloured, but cytoplasm has little 

 tendency to take up the lake. As a result the exact period of dyeing 

 is unimportant. These dyes are particularly suitable for whole 

 mounts. Since too strong coloration does not occur easily, the 

 outer parts of a piece of tissue are not dyed very differently from 

 the interior. 



It is interesting to notice the relative amounts of metal and dye 

 in such fluids as these. The weight of potassium alum crystals that 

 contains one atomic weight in grams of aluminium is almost 

 exactly equal to one gram molecular weight of carmine. Therefore, 

 if one wished to have a solution with 3 times as many dye 

 molecules as aluminium atoms, so as to combine as much dye as is 

 theoretically possible with the metal, one would take 3 g of car- 



