NUCLEIC ACIDS IN CHROMOSOMES AND MITOTIC DIVISION 185 



2. Analysis of Chromosomes in Situ 



a. The Feulgen Reaction 



The chromosomes can be visualized in situ by their abiUty to combine 

 with specific dyes. vSince the work of Feulgen and Rossenbeck,^ the method 

 most commonly utilized for demonstrating the chromosomal DNA has been 

 the Feulgen nucleal reaction, which is discussed in detail in Chapter 17. 



The exact course of the Feulgen reaction is not yet fully understood, but it is gen- 

 erally assumed that a liberation of aldehyde groups from the DNA takes 

 place through partial acid hydrolysis and that these aldehyde groups react with 

 leuco-basic fuchsin (the Schiff reagent) forming a red-colored product. This reac- 

 tion has also been used for the microchemical determination of DNA in amounts 

 between 0.5 and 2 mg. but not in the presence of proteins.'* ■'' Since the reaction does 

 not proceed stoichiometrically, a DNA standard always must be used at the same 

 time. 



Nevertheless, the Feulgen nucleal reaction has been used by several authors for 

 the quantitative determination of DNA in histological preparations." Since, in nuclei 

 with a DNA concentration up to a few per cent, the intensity of the color developed 

 is proportional to the DNA content, '^ the relative amounts of DNA per nucleus in 

 a cell population can in some cases be estimated, ^"-^^ and the results expressed in 

 arbitrary units. 



There has been considerable controversy concerning the specificity of the Feulgen 

 reaction in cytological preparations, but it is generally agreed at the present time 

 that common biological materials contain little or no material other than DNA that 

 can give a proper nucleal reaction. Whether DNA, with or without the recolorized 

 leuco-basic fuchsin, is able to diffuse throughout the cell so as to give a false picture 

 of localization has also been discussed."" Partially hydrolyzed DNA from which 

 only purine bases have been split off,^* is however insoluble in weak acid and reacts 

 with the Schiff reagent to give the characteristic bluish-red color. This color is de- 

 veloped on the insoluble material, the solution above it remaining colorless. 



Thus the most important conclusion from the numerous investigations 

 carried out by means of the Feulgen nuclear reaction can be regarded as 

 valid, namely, that the DNA in the cell is in general concentrated in the 

 chromosomes. This conclusion holds for material after acid hydrolysis and 



'« G. Widstrom, Biochem. Z. 199, 298 (1928). 



'^ T. Caspersson, Biochem. Z. 253, 97 (1932). 



18 H. S. di Stefano, Chromosoma 3, 282 (1948). 



'9 H. Ris and A. Mirsky, /. Gen. Physiol. 33, 125 (1949). 



2° R. E. Stowell, /. Natl. Cancer Inst. 3, 111 (1942). 



21 A.W.PoUisier a.ndR.Ris, Cold Spring Harbor SymposiaQuant. Biol. 12, 147 (1947). 



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



" C. Vendrely, Bull. biol. France et Belg. 84, 1 (1952) ; see also A Pollister, M. Himes, 



and L. Ornstein, Federation Proc. 10, 629 (1951). 

 2" E. Stedman and Ellen Stedman, Symposia Soc. Exptl. Biol. 1, 232 (1947). 

 " H. N. Barber and H. G. Callan, Nature 153, 109 (1949) ; see also J. O. Ely, and M. H. 



Ross, Anal. Record 194, 103 (1949). 

 " C. H. Li and M. Stacey, Nature 163, 538 (1949); W. G. Overend and M. Stacey, 



ibid. 163, 538 (1949). 



