Feulgen Densitometry: Importance of a Stringent Hydrolysis Regime

Abstract

Abstract: Feulgen densitometry is still a widely used method for DNA content measurements, but experimental procedures and results are often controversial. The present note is concerned with a recent report in the literature that optimum Feulgen staining required a remarkably longer hydrolysis time with 5 M HCI in Dactylis glomerata L. than in Hordeum vulgare L. (i.e., 62 min versus 20 min at 25 d̀C). As this result is prone to question the usual practice of maintaining unified hydrolysis times for test material and internal standard, we established hydrolysis curves for D. glomerata, H. vulgare, Pisum sativum L. and Allium cepa L. at 20 d̀C and 25d̀C for 0 to 100 min. No striking differences between the species and, in particular, between Doctylis and Hordeum were found. Optimum staining occurred after 60 min with hydrolysis at 20 d̀C and after 25 min at 25 d̀C. It is strongly recommended to conduct the quantitative Feulgen reaction at a precisely controlled temperature instead of an inexact room temperature. The broader plateau of optimum staining at 20 d̀C makes this regime preferable.