Microspectrophotometric analysis of the effects of various hydrolysis times and fixatives on the intensity of the Feulgen reaction on rat liver and slime mold myxamoebae

Abstract

The two wavelength method of microspectrophotometry was used to analyze the effects of various hydrolysis times for the four different fixatives (Calcium-acetate-formalin, Acetic-alcohol-formalin, Neutral buffered formaldehyde and Fixcel - a new fixative); to determine the best hydrolysis time for each fixation procedure that will give maximal intensity and quantification of the Feulgen reaction for both tissue types (liver and myxamoebae), and therefore most types of tissues. Optimum Feulgen staining intensity for both tissue types was obtained with Calcium-acetate-formalin (CAF) as a fixative when compared to the other three fixatives. Liver and myxamoebae tissue fixation with Acetic-alcohol-formalin (AAF) and fixcel attained maximum Feulgen intensity rapidly. Liver tissue fixation with Neutral buffered formalin exhibited the longest plateau period of maximal Feulgen intensity, and in myxamoebal tissue fixation with AAF exhibited the longest extended plateau period of maximal Feulgen intensity reflecting tissue differences.

The hydrolysis curve for liver tissue fixed in CAF exhibited an abrupt decline in maximal Feulgen staining intensity following a 5 minute plateau period. Such an abrupt decline was not evidenced in the descending slope of the hydrolysis curve for myxamoebal nuclei fixed in CAF, nor in comparison with other fixatives. The results of this investigation indicate that fixcel is a very poor fixative for liver tissue, but myxamoebal tissue fixed in fixcel suggests that it may be used in certain tissue types for quantification of DNA; furthermore, similar Feulgen intensities should not be expected to occur between two cell types when using the same fixatives.