58 HEWSON SWIFT 



As shown by many workers,^*-" an increase in dye concentration will in- 

 crease the amount of dye bound, and extend the pH range where appre- 

 ciable binding occurs. This is also shown in Fig. 1. An increase in the con- 

 centration of buffer ions may be expected either to decrease or increase 

 binding depending on the nature of the anions and cations used. As men- 

 tioned above, all ions may be expected to compete to some extent with 

 dyes of like charge, but competition is usually greater with larger buffer 

 molecules.-^ Also, as discussed by Danielli,^^ the pH near protein surfaces 

 may be markedly different from that in the dye solution, and the difference 

 is accentuated by increased ionic strength. The effect of salts on dye bind- 

 ing has also been discussed by Neale.^^ 



3. Fixation 



Several factors are obviously of importance in determining the effects of 

 fixation on dye binding. Certain tissue constituents may be soluble in the 

 fixative, and be removed, at least in part, by it. For example, some tissue 

 proteins are apparently not immediately precipitated by 10% neutral 

 formalin, and are removed from cells in smears, or from the outer portion 

 of tissue blocks. Fixative may also alter electrostatic charges on nucleo- 

 protein complexes by combining with ionizable groups. For example, for- 

 malin is known to combine with amino groups, and the metal cations in 

 many fixatives, such as mercury and chromium, may be strongly bound by 

 carboxyl and phosphoryl groups. Because the effects of complicated histo- 

 logical fixatives are obviously highly involved and often unpredictable, 

 many workers have preferred to use simple fixatives, such as acetic alcohol 

 or freeze-drying followed by alcohol treatment. Enzymes (deoxyribo- 

 nuclease and ribonuclease) work w^ell after such fixatives, and acid extraction 

 of nucleic acids is also readily performed. Brachet^^ has found dye binding 

 to be somewhat depressed after freeze-drying, however, possibly because 

 of hpid interference. 



4. Affinity 



Methyl green shows, under certain special conditions, an affinity for 

 DNA but not for PNA."'^"-^^ From the in vitro studies discussed above, it 

 is evident that more methyl green is bound to highly polymerized than to 

 depolymerized nucleic acid. The conditions under which methyl green 

 stains DNA in tissue sections thus appear to be those in which the equi- 

 librium factors are adjusted so that the affinity for the lower polymer PNA 



" N. D. Levine, Stain Technol. 15, 91 (1940). 



38 J. F. Danielli, Biochem. J. 35, 470 (1941). 



39 S. M. Neale, Trans. Faraday Soc. 42, 473 (1946). 



*" N. B. Kurnick, Cold Spring Harbor Symposia Quant. Biol. 12, 141 (1947). 

 ^' C. Leuchtenberger, Chromosoma 3, 499 (1950). 



