CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 61 



bond strength of the dye-nucleate, with consequent rapid or slow dye 

 extraction. 



One aspect of differentiation, much used by classical cytologists, is that 

 of controlled "destaining." As mentioned below, more stain may be bound 

 to structures with high nucleic acid concentrations, such as mitotic chromo- 

 somes and pycnotic or lymphocyte nuclei. These structures may have larger 

 electrostatic fields, possibly because the ratio between available nucleic 

 acid phosphoryl and protein basic groups is different than in the surround- 

 ing areas. In addition to binding more dye, such regions also apparently 

 lose dye less rapidly, so that by using conditions causing heavy staining 

 followed by controlled "destaining" as is done, for example, with iron 

 hematoxylin, a point is reached where these structures alone are densely 

 colored with a weakly staining background. By proper manipulation, 

 "differential staining" of mitotic chromosomes is possible with a variety of 

 basic dyes. In some cases factors other than electrostatic forces may also 

 be involved, such as cell permeability to the dye-iodine complex in the 

 Gram reaction.*^ 



6. Metachromasy 



Many basic dyes stain certain cell constituents one color and others 

 another. This characteristic has been studied particularly by Lison-^ and 

 Michaelis.^^'^" It deserves brief mention here because, as shown by Flax and 

 Himes,^^ it affords a very simple way of differentiating PNA and DNA in 

 tissues, and also may, when the phenomenon is better understood, provide 

 some information on the arrangement of stainable groups in the nucleic 

 acid molecule. Many basic dyes in solution do not show a linear rela- 

 tionship between concentration and optical density (Beer's law). With 

 increasing concentration the color of the dye shifts and one or two new ab- 

 sorption peaks appear on the shorter wavelength side of the original absorp- 

 tion maximum. Several workers^^'^'*^ have suggested that the three peaks 

 represent dye monomers, dimers, and higher polymers, respectively, formed 

 through Van der Waals interactions. In tissues, the polymer peaks are par- 

 ticularly characteristic of the sulfuric ester binding of mucin and chon- 

 droitin as pointed out by Lison.-^ Such substances stain red-purple with 

 toluidine blue, w^hile nucleic acids stain blue. Bank and Bungenberg de 

 Jong^^ attributed the red staining to areas of higher charge density. Differ- 

 ences in the color of staining in some cases afford an excellent way to 



^^ T. Mittwer, J. W. Bartholomew, and B. J. Kallman, Stain Technol. 25, 169 (1950). 



5" L. Michaelis, J. Phys. & Colloid Chem. 54, 1 (1950). 



" E. Rabinowitch and L. Epstein, J. Am. Chem. Soc. 63, 69 (1941). 



" S. E. Sheppard, Revs. Mod. Phys. 14, 303 (1942). 



" 0. Bank and H. G. Bungenberg de Jong, Protoplasma 32, 489 (1939). 



