Feulgen Densitometry: Importance of a Stringent Hydrolysis Regime

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 HCl in Dactylis glomerata L. than in Hordeum vulgare L. (i.e., 62 min versus 20 min at 25°C). 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°C and 25°C for 0 to 100 min. No striking differences between the species and, in particular, between Dactylis and Hordeum were found. Optimum staining occurred after 60 min with hydrolysis at 20°C and after 25 min at 25°C. 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°C makes this regime preferable.

Key words

Densitometry - Feulgen - hydrolysis - Dactylis glomerata - Hordeum vulgare