70 Microscopic Histochemistry 



common property of these dyes is that their shade depends 

 on the concentration of the solution (although this feature 

 has not been investigated in the case of methyl violet ) . Dilute 

 solutions are bluish, and, as the concentration is raised, the 

 shade shifts progressively toward red. The absorption spectra 

 of these dyes show two ( or three ) maxima. In the case of to- 

 luidin blue, for instance, one of the peaks ( a ) is around 630 

 m^, the second one ( /5 ) around 590 m^u,, and the third one 

 (y) around 560-70 m^u,. In dilute solutions the a peak is the 

 tallest; the other two are barely noticeable. With increasing 

 dye concentration the /3 peak will become more and more 

 prominent and actually surpass the a peak. The y peak is 

 observed only at rather high concentrations. Acidification, 

 high temperature, and the addition of alcohol tend to sup- 

 press the y8 peak; alkalinization, low temperature, and the 

 addition of chromotropic substances have the opposite effect. 

 The physicochemical nature of metachromasia is not com- 

 pletely elucidated. According to Lison, ^^' ^^ it is due to a 

 tautomeric form ( imino base ) of the dye, which is in a labile 

 equilibrium with the orthochromatic form. This equilibrium 

 is displaced toward the metachromatic form by chromo- 

 tropic substances. Michaelis and Granick,^^ on the other 

 hand, have formulated the theory that the metachromatic 

 form of the dye is simply a dimer ( or polymer ) of the ortho- 

 chromatic (monomer) form. In the absorption spectrum, the 

 absorption maximum of the monomer is represented by the 

 a peak; that of the dimer by the /3 peak; that of polymers 

 by the rather flat plateau of the y peak. Factors favoring 

 polymerization are high concentration, low temperature, rela- 

 tively high pH values, an aqueous medium, and especially 

 the presence of large molecules with suitably placed and 

 spaced acidic groupings (-COOH, -OPO(OH)2, -OSO2OH) 

 which can hold the polymer dye molecule together by its 



59. Lison, L.: Acad. roy. Belgique, cl. de sc, 19:1332, 1933. 



60. Lison, L.: Arch, de biol., 46:599, 1935. 



61. Michaelis, L., and Granick, S.: J. Am. Chem. See, 67:1212, 1945. 



