Quantitative aspects of the cytochemical Feulgen-DNA procedure studied on model systems and cell nuclei

Abstract

Quantitative aspects of DNA losses during fixation and pararosaniline(SO2)-Feulgen staining of microscopic preparations were studied. The preparation of a new cytochemical model, consisting of DNA-protein layers (with thicknesses between 0.1 and 5.0 micrometer) on microscopic glass slides is described and potentialities and limitations of this model are discussed. Polyacrylamide films into which high molecular weight calf thymus DNA or chicken erythrocyte nuclei had been constrained served as another model. As biological objects chicken erythrocyte nuclei and rat liver nuclei either in suspension or on microscopical glass slides were used. The experimental results indicate a loss of about 5% of the DNA due to the fixation procedure applied. Hydrolysis in 5 N HCl at room temperature, staining with the pararosaniline-Schiff medium and rinsing with sulfurous acid induced losses of DNA too, varying in amount depending on the type of preparation used. About 10% of the original DNA content is lost in total from chicken erythrocyte nuclei and rat liver nuclei dried on microscopical glass slides, from chicken erythrocyte nuclei constrained in polyacrylamide films, and from DNA-protein layers on microscopic glass slides. For nuclei fixed and stained in suspension the total losses amount to about 40%. The differences in losses between various types of preparations are discussed. Biochemically, the content of DNA originally present per chicken erythrocyte nucleus was determined to be 2.52 pg, a value, which is in good accordance with reliable biochemical data published already. It is shown that calibration of cytochemical staining intensities into biochemical units or absolute amounts of material by use of a model system, is only reliable when it is known or to be expected that both the loss of material due to fixation and staining, and the stoichiometric relation between material present and dye molecules is identical. The same holds for the application of internal biological reference systems.

Distribution of DNA, nuclear micro-heterogeneities and compaction of the chromatin in rabbit epididymal spermatozoa. Ultrastructural evaluation of the Feulgen-like technique using osmium ammine

“…At the ultrastructural level, the Feulgen-like procedure of Cogliati and Gautier (1973) was first used by Biggiogera (1986 and1989) to show micro-heterogeneities in mouse spermatozoa. These data were further confirmed by Courtens et al (1991 ) (Biggiogera, 1989); and (ii) the sperm chromatin are more affected by hydrolysis than the somatic chromatin (van Prooijen-Knegt et al, 1980;Redi et al, 1982Redi et al, , 1986, and some of the DNA is released from the samples (Silva and Mello, 1986).…”

Evidence for haploidy in metacyclic forms of Trypanosoma brucei.

“…Although this possibility cannot be completely ruled out, we think it is unlikely because it is not supported by any available evidence. In particular, no known factor in the DNA structure, conformation, or compactness can account for a 2-fold difference in the Feulgen-reaction fluorescence intensity (8,(15)(16)(17). In addition, the microscopic examination of the chromatin in metacyclic trypanosomes shows no trace of pycnosis or of any other feature that could account for this difference in fluorescence.…”

“…These forms are found in the saliva of flies just before and after development of the parasite in the salivary glands, respectively. The quantitative cytochemical method we used is based on the observation of a linear relationship between the amount of fluorescence and the DNA content of Feulgen-stained nuclei under appropriate fixation, hydrolysis, and staining-procedure conditions (8)(9)(10).…”

Evidence for diploidy in metacyclic forms of African trypanosomes.

“…It is based on the covalent binding ofthe fluorescent dye to the aldehyde groups generated on the backbone of the DNA by hydrolytic removal of the purine bases. Under defined hydrolysis and staining conditions, binding is stoichiometric and the staining reaction is highly specific for DNA (23). Histograms were analyzed as indicated in the legend of Fig.…”