A CRITIQUE OF CYTOCHEMICAL METHODS 215 



ill which there was nearly negative cytoplasmic basophilia in cells which, 

 by ultraviolet absorption, were shown to contain considerable nucleic acid 

 (Pollister . . . Breakstone, 1951). In view of these difficulties it is per- 

 haps best to regard basophilia as a useful indicator of sites of major nucleic 

 acid concentration, which then becomes a guide to application of more 

 satisfactory (lualitative and c^uantitative methods. In fact, this has been 

 the role of l)asophilia in development of modern concepts of the intracel- 

 lular distribution of nucleic acid. Although the methods of identification 

 by the ultraviolet absorption spectrum measure nucleic acid directly and 

 are therefore potentially applicable to situations where polynucleotide 

 basophilia might be misleading, for the most part ultraviolet studies have 

 proceeded along lines which were clearly foreshadowed by old findings of 

 the basophilic reactions of cells. This is strikingly emphasized also by 

 the fact that practically all the fundamental conclusions about intracellu- 

 lar distribution of nucleic acids which the Caspersson school reached by 

 use of ultraviolet absorption techniques and used in elaborating compre- 

 hensive theories of cell function (see Caspersson, 1950) were arrived at 

 independently by Brachet and his collaborators (Braehet, 1944) with 

 only nuclease-digestible basophilia as a guide. Indeed, since Brachet and 

 coworkers used nucleases in combination with basophilia, they were able 

 to detect ribonucleic acid in chromatin, while it was necessarily overlooked 

 in the less specific ultraviolet absorption studies. 



There are certain applications of basophilia to (lualitative cytochem- 

 istry which are of special interest because not only do they demonstrate 

 the presence of strongly acidic substances, but also by specific color 

 changes they appear to indicate something of the intramolecular structure 

 of the acidic substrate with which they combine. The best known of 

 these is the so-called "metachromatic" basophilia (metachromasia) by 

 which certain cellular structures stain red with dyes which appear blue in 

 solution (e.g., toluidine blue and azure). This method was empirically 

 recognized long ago (Ehrlich, 1877; Hoyer, 1890). It has been the sub- 

 ject of a number of chemical and spectrophotometric studies (e.g., Kelley 

 and Miller, 1935a, b; Lison, 1935; Bank and Bungenberg de Jong, 1939; 

 Wislocki et al., 1947; Michaelis and Granick, 1945). Spectrophotometric 

 analysis shows that, whenever these dyes are in water solution, three 

 states are in ecjuilibrium. An a absorption peak in the red part of the 

 spectrum represents unaggregated dye in the "monomeric" state; a 

 absorption peak (green) is believed to represent dye in the two-molecule 

 aggregate, or "dimer," state. A /x absorption peak (in the blue-green) 

 supposedly represents highei- states of aggregation than the dimer which 

 may for conv^enience be called a "polymer" state. The aggregation, as 

 would be expected, is dependent on concentration, with the result that 

 these basic dyes notably fail to follow Beer's law. In stained cells, basic 

 dyes are, of course, removed from solution and combined with the solid 



