CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 59 



is negligible, but that for DNA is strong enough to cause an appreciable 

 binding. If the equilibrium is shifted toward an increased nucleic acid 

 binding, however, the "specificity" breaks down, and both DNA and PNA 

 stain. This shift may be brought about by blocking amino groups through 

 strong formalin fixation,^^ ^y acetic anhydride, by removal of amino groups 

 with nitrous acid,^^ or by increasing the pH of staining to 5.0 or above. It 

 is obvious that methyl green may be used as a "specific" stain for DNA 

 only where the factors determining the dye equilibrium are carefully con- 

 trolled. 



As shown by Unna,"*^ treatment of slides for 10 minutes with water at 

 90° C. will abolish methyl green staining although the Feulgen reaction is 

 still strong. Kurnick^^ postulated that the loss of staining was due to de- 

 polymerization of the DNA, and Pollister and Leuchtenberger^^ suggested 

 that DNA stainability, when compared with the Feulgen reaction, might 

 be an indicator of the degree of polymerization or at least of the steric con- 

 dition of the DNA in tissues. This conclusion now seems unlikely for several 

 reasons. (1) True depolymerization as caused by treatment with deoxy- 

 ribonuclease results in removal of DNA from the slide. Appreciable DNA 

 removal does not occur in the 15 minutes necessary to inhibit methyl green 

 binding. (2) Heat depolymerization of DNA in solution as studied by 

 Kurnick'^ took longer to complete, and then resulted in a decrease in dye 

 binding of only 60 to 70 %. The loss of stain in tissue is complete with much 

 shorter treatment. (3) The DNA stainability may be brought back after 

 hot water treatment by blocking or removal of the protein amino groups.'* 

 None of these arguments completely disproves the possibility that some 

 slight depolymerization or steric change may occur and that the reduced 

 affinity of methyl green is an indication of it. A more plausible theory, 

 however, is that the hot water possibly releases more amino groups to bind 

 DNA phosphoryl groups, or at least to alter the charge in their vicinity, and 

 thereby reduces the availability of phosphoryl groups to the dye. A slight 

 reduction in azure B binding was reported by Alfert'* after similar hot 

 water treatment, and a slight decrease in toluidine blue binding in vitro was 

 reported upon heat treatment of a PNA fibrinogen mixture by Herrman, 

 Nicholas, and Boricious.^ Although the variations in methyl green binding 

 that have been reported in various tissues'*^ '^^ •*® are of interest, their inter- 

 pretation at present is obscure, and may merely involve alterations in the 

 amount or nature of adjacent proteins. 



Although all basic dyes will show binding with nucleic acids in tissue 



« H. Swift, Physiol. Zool. 23, 169 (1950). 



« P. G. Unna, Monatsh. prakt. Dermatol. 62, (1887). 



'* A. W. Pollister and C. Leuchtenberger, Proc. Natl. Acad. Sci. U.S. 35, 111 (1949). 



« P. B. Weisz, /. Morphol. 87, 275 (1950). 



« N. J. Harrington and R. W. Koza, Biol. Bull. 101, 138 (1951). 



