NUCLEOCYTOPLASMIC RATIO 



240 



NUCLEOCYTOPLASMIC RATIO 



wide variety of basic dyes, both in the 

 test tube and in tissue sections. In 

 a rougli way, it can be stated that the 

 amount of a basic dye taken up by a 

 given nucleic acid-containing structure 

 in the cell is proportional to the amount 

 of nucleic acid present. Attempts at 

 a precise quantitative estimation of 

 nucleic acid content by means of basic 

 dye uptake are in the early stages of 

 development. 



The physico-chemical nature of the 

 interaction, in buffered aqueous solu- 

 tion, of rosaniline with yeast and 

 pancreatic pentose nucleic acids (PNA) 

 and with thymus desoxypentose nucleic 

 acid (DNA) has recently been studied 

 (Cavalieri, L. F., Kerr, S. E., and 

 Angelos, A. J. Am. Chem. Soc. 1951, 

 73, 2567-2578; Cavalieri, L. F. and 

 Angelos, A., ibid., 1951,72, 4686-4693). 

 The reaction was found to be reversible 

 and stoichiometric . The curves relating 

 amount of dye taken up by the nucleic 

 acid to the concentration of free dye 

 were different for each type of nucleic 

 acid. However, in all cases the data 

 could be explained satisfactorily on the 

 basis of a salt-like linkage between the 

 dj^e cation and the negatively charged 

 phosphoric acid groups of the nucleic 

 acid, without the assumption of more 

 complicated adsorptive forces. Speci- 

 fic dissociation constants for the dye- 

 nucleic acid complexes could thus be 

 calculated. Similar studies using other 

 common histologic dyes are needed 

 before the staining of nucleic acids in 

 tissue sections can be quantitatively 

 evaluated. It should be noted that 

 the dye concentrations used in the 

 above experiments were far below those 

 ordinarily employed in staining. There 

 is some evidence, however, that the dye- 

 nucleic acid interaction is stoichio- 

 metric at high dye concentrations also. 



Mixtures of methyl green and pyronin 

 stain nuclei green and cytoplasmic 

 nucleoproteins red (see Methyl Green- 

 Pyronin). This differential staining of 

 the two types of nucleic acid by two 

 different basic dyes has been investi- 

 gated by N. B. Kurnick (J. Gen. Phys- 

 iol., 1950, 33, 243-264), who concluded 

 the DNA of the nucleus owed its specific 

 staining by methyl green to its high 

 degree of polymerization. DNA which 

 had been depolymerized by treatment 

 with heat or acid failed to stain well 

 with methyl green but did stain strongly 

 with pyronin or other basic dyes. DNA 

 combined with histone stained less 

 strongly with methyl green than did the 

 free DNA. Under conditions of high 

 dye concentrations, the reaction be- 

 tween DNA and methyl green was said 



to be stoichiometric (Kurnick, N. B. 

 and Mirsky, A. E., J. Gen. Physiol., 

 1950, 33, 265-274), and has been made 

 the basis of a quantitative histochem- 

 ical test for DNA (Leuchtenberger, C, 

 Vendreley, R., and Vendreley, C, Proc. 

 Nat'l. Acad. Sci. 1951, 37, 33-38). 



The interaction of nucleic acids with 

 basic dyes, while apparently primarily 

 of an ionic character, can be modified 

 by many factors. Among these are: 

 1) availability of the phosphate groups 

 of the nucleic acid and their spatial 

 arrangements (polymerization) ; 2) com- 

 petition for available phosphate groups 

 by substances other than dye, such as 

 the histones of the nucleus, heavy metal 

 cations of the fixing fluids, or impurities 

 in the dye solution; 3) ionic strength, 

 pH, and temperature of the staining 

 bath; and 4) the dye concentration used 

 for staining. To approach any degree 

 of stoichiometry in dye-nucleic acid 

 interactions, all of these factors must 

 be controlled as rigidly as possible. 



The absorption spectra of all basic 

 dyes which have been investigated show 

 changes when the dyes are bound to 

 nucleic acids. This fact is of im- 

 portance if the measured absorption at 

 some particular wave length is to be 

 used to measure the amount of dye pres- 

 ent in a cellular structure. It may 

 also provide information as to the 

 structure of the nucleic acids them- 

 selves. Such spectral shifts have been 

 studied by Michaelis and Granick 

 (Michaelis, L., J. Phys. and Colloid 

 Chem. 1950, 54, 1-17), who found that 

 basic dyes bound to nucleic acids almost 

 invariably show a shift in their absorp- 

 tion maxima towards the longer wave 

 lengths. Binding to nucleic acid was 

 also found to inhibit the development 

 of metachromasia shown by such dyes 

 as thionin and toluidin blue either in 

 concentrated solutions or when bound 

 to other strongly acid substrates such 

 as the acid mucopolysaccharides. 

 Under conditions of excess nucleic acid 

 with regard to the dye, the bound dye 

 was shown to follow the Beer-Lambert 

 Law. Under conditions of high dye 

 concentration with regard to nucleic 

 acid, such as obtain in the usual stain- 

 ing reactions, it has not been shown 

 that the bound dye follows Beer's law. 

 Nucleocytoplasmic Ratio. A histological 

 method for computing this ratio is fully 

 described by Cowdry, E. V.and Paletta, 

 F. X., J. Nat. Cancer Inst., 1941, 1, 

 745-759 ; but there are many such tech- 

 niques. A chemical method has been 

 used to advantage by Dawbarn, M. C, 

 Australian J. Exp. Biol. & Med. Sci., 

 1932, 9, 213-226. Her ratio is obtained 



