Feulgen Cytophotometry of Pine Nuclei; Effects of Fixation, Role of Formaline

Abstract

The effects of 5 fixatives: FAA, [formalin, acetic acid, ethanol] Carnoy''s, [acetic acid, ethanol, chloroform] Craf III, [chromic acid, acetic acid, formalin] formalin and glutaralde-hyde were analyzed for use in quantitative Feulgen cytophotometry of pine embryo tissues. Craf III and glutaraldehyde had serious deficiencies because they depressed the absorption peak, severely interfered with DNA extraction and in the case of glutaraldehyde there was considerable cytoplasmic dye-binding. Neutral 10%formalin gave good tissue fixation but did not permit the degree of enzymatic or acid extraction of DNA as did Carnoy''s solution. Haupt''s adhesive, with the usual 4% formalin as a hardener, at temperatures of 45-56 C completely prevented the enzymatic extraction of nuclear DNA by DNase and also greatly increased the resistance of the DNA to mineral acid hydrolysis. Denaturation of DNA by formalin appeared to be responsible for these results. Absorption was linearly related to both section thickness and DNA concentration per nucleus.