74 HEWSON SWIFT 



ables has recently been studied by several workers." '^^-'i- Although con- 

 stituents may be varied over rather wide limits and still produce a service- 

 able Feulgen reaction, for quantitative studies some control over the 

 composition of the reagent is desirable. Most quantitative determinations 

 on the sensitivity of the reagent have been made on formaldehyde or DNA 

 solutions. The situation for nuclei may not be strictly comparable. More 

 measurements on tissue sections after various treatments would be helpful. 

 As one would expect if recolorization proceeds according to Wieland and 

 Scheuing," excess SO2 is needed in the reagent solution. With formalin 

 solutions, Atkinson" found a concentration of 10 moles of bisulfite to 1 of 

 dye was optimal, with a decrease in intensity with smaller or larger bisulfite 

 concentrations. For DNA solutions, Ely and Ross*^ obtained an increasing 

 intensity with metabisulfite concentration, and no optimal concentration 

 was reached even when molar ratios were very much higher. This is an im- 

 portant variable, since the SO 2 content of the reagent readily changes 

 through evaporation (Table II). The pH of the reagent solution also affects 

 the intensity of the reaction, as shown in Table II. A reduction in color 

 with decreasing pH has been described for formaldehyde"^ and DNA^^ 

 solutions. 



7. Quantitative Aspects 



The relation between amounts of DNA and regenerated dye intensity in 

 vitro was studied by Caspersson,i°^ who concluded that estimates could be 

 made to 2% provided proteins were not present in appreciable amounts. 

 Rumpf"^ concluded that the Schiff reagent was not quantitative for alde- 

 hydes, although Atkinson" found a linear relation for formaldehyde. 

 Lessler*'' used drops of DNA-gelatin mixtures on slides to test the linearity 

 of color intensity, and found it was proportional to the DNA concentration 

 only up to 1 mg. of DNA per miUiliter of 20% gelatin. Above that concen- 

 tration the intensity was depressed. The presence of histone under some 

 conditions was found to enhance and under other conditions to depress the 

 dye intensity."^ '"^ Also the intensity decreases in the SO2-HCI rinse solu- 

 tions"" at a rate dependent on their composition."^ 



In a few cases the amounts of DNA per nucleus, as determined bio- 

 chemically, have been compared with the intensity of Feulgen-stained 

 nuclei as measured with a microphotometer. The determinations made by 

 Ris and Mirsky"^ with the two methods matched to within 10%. In each 



"2 J. B. Longley, Stain Technol. 27, 161 (1952). 



"3 P. Rumpf, Ann. chim. (Pans) [11]3, 327 (1935). 



"^ A. Shibatani, Naiuie 166, 355 (1950). 



'15 H. Naora, H. Matsuda, M. Fukuda, and A. Sibatani, /. Japan. Chetn. 5, 729 (1951). 



'18 A. Shibatani, Experientia 8, 268, 1952. 



^" H. Ris and A. E. Mirsky, J. Gen. Physiol. 33, 125 (1949). 



