A Chromatographic Study of Hydrolysis in the Feulgen Nucleal Reaction* by Philip

Acid hydrolysis of tissue has long been recognized as a necessary part of the Feulgen method for demonstrating the presence of DNA (deoxyribonucleic acid) in histological preparations (Feulgen and Rossenbeck, 1924). When attempts were made to use the method for the quantitative determination of DNA (Pollister and Ris, 1947; Di Stefano, 1948; Ris and Mirsky, 1949; Swift, 1950 a), the role of hydrolysis became especially significant. In spite of numerous studies of the Feulgen reaction the effects of hydrolysis on DNA are still not fully understood. The study of hydrolysis in the Feulgen procedure has been seriously limited because of lack of suitable methods of analysis. Recently, biochemical techniques have been devised which permit qualitative and quantitative studies on the purines and pyrimidines of tissue nucleic acids (Marshak and Vogel, 1951; Wyatt, 1951). These methods consist of extraction of nucleic acids from tissue, digestion to the free bases, and separation by paper chromatography. In the present study on hydrolysis of fixed tissue sections, certain of these chromatographic techniques were used. At various times during hydrolysis, hydrolysates were analyzed for some of the degradation products of nucleic acid. At corresponding times similar sections were stained with Feulgen reagent, and the Feulgen dye content per nucleus was measured with a microphotometer. In this way it was possible to determine the course of events as DNA was hydrolyzed in the Feulgen procedure. Since thymine is characteristic of DNA and uracil of RNA (ribonucleic acid) the presence of degradation products containing these bases was used to indicate the destruction of the two types of nucleic acid.