Quantitative cytophotometric analysis of the effects of various fixatives and tissue types on hydrolysis times and staining intensities using the blue feulgen reaction

Abstract

Three fixatives, ten percent buffered formalin, Carnoy's fixative, and a new fixative, Streck's Tissue Fixative (STF), were used to fix four tissue types - murine liver and sperm, myxomycete plasmodium and myxamoeba-- to determine their influence on hydrolysis times and staining intensities in the Blue Feulgen reaction. Relative amounts of DNA per nucleus were determined using the two wavelength method of quantitative microspectrophotometry. Liver fixed in formalin and Carnoy's fixative exhibited similar peak hydrolysis plateaus, while the hydrolysis plateau for STF-fixed liver cells began later. Liver fixed in all three fixatives exhibited more intense staining after 60 minutes in the stoichiometric cresyl violet stain than after 20 minutes, so all tissues were stained for 60 minutes. All three fixatives proved to be effective for use with the Blue Feulgen reaction, as all three provided approximate 2:1 ratios between DNA content of the diploid tissues (liver and plasmodium) and their respective haploid tissues (sperm and myxamoeba). Carnoy's fixative, however, is not recommended for use with the Blue Feulgen reaction because it provided lower stain intensities for all four tissues. The alcohol-based Carnoy's fixative is known to extract some Feulgen-stainable material. Ten percent buffered formalin and STF provided excellent staining, with STF exhibiting 10 to 20 percent stain enhancement over formalin or Carnoy's fixative for all four tissue types. The new fixative, STF, also exhibited great sensitivity to changes in DNA content during the cell cycle and is highly recommended for use with Blue Feulgen microspectrophotometry.