The effects of various hydrolysis times and fixation on the intensity of the Feulgen reaction in studies involving quantitative microspectrophotometry

Abstract

The effect of three different fixatives, ten percent buffered formalin, Lillies alcohol-acetic acid-formalin, and a new fixative, bicarbonate formaldehyde, on the staining intensity of the Feulegen nucleal reaction for both liver and myxamoebal nuclei was analyzed by means of the two wavelength method of microspectrophotometry. Comparisons of the relative DNA measurements of each procedure were made to determine the reaction parameters best suited to each tissue type in providing maximal straining intensity values.

Fixation of the liver nuclei in bicarbonate formaldehyde provided the best maximal staining intensity of all three fixatives, while fixation in ten percent buffered formalin yielded the best maximal intensity for myxamoebal nuclei. Identical maximal stain intensity values were obtained for amoebal nuclei fixed in both Lillies AAF and bicarbonate formaldehyde with the hydrolysis curves for each exhibiting extended plateau periods as opposed to a significant decline in plateau in fixation with ten percent buffered formalin. An extended period of hydrolysis before attainment of maximal stain intensity characterized the effect of Lillies AAF on liver nuclei, while fixation in ten percent buffered formalin resulted in an extremely prolonged plateau period and in early attainment of maximal intensity, as was the case with bicarbonate formaldehyde fixation. The maximal stain intensity values for fixation of liver nuclei in ten percent buffered formalin and Lillies AAF were closely related and fell well below the value obtained with fixation in bicarbonate formaldehyde. In the case of both liver and myxamoebal nuclei, fixation with Lillies AAF resulted in extreme nuclear and cytoplasmic shrinkage and should be regarded as relatively harsh fixation procedure on tissue preparations.

Statistical analysis of the mean relative DNA values representing the periods of maximal stain intensity of the three fixative for each cell type indicated that no significant differences exist between these values. Therefore, each procedure can provide both a stoichiometric relationship and valid comparable values of relative amounts of DNA in view of the specific effect each fixative has in determining the resultant configuration of the nucleic acid for dye-binding.

It is evident from these experiments that the fixative and hydrolysis times selected for the performance of a valid and quantitative Feulgen DNA determination must be chosen properly for each specific tissue type since differences in staining intensity may occur.