[Effect of the hydrolyzing solution temperature, acid concentration and the duration of the hydrolysis on the intensity of the Feulgen reaction]

Abstract

The quantity of DNA-fuchsin in the cock monocytes and erythrocytes has been measured by scanning cytophotometric methods and computer analysis. Hydrolysis was carried out using 1 N HCl (60 degrees C, 4-30 min) and 5 N HCl (37 degrees C, 4-36 min, and 22 degrees C, 10-150 min). The elevation of the temperature from 22 degrees to 37 degrees resulted in a 5-fold reducing of the time required for achieving the maximum of the hydrolysis curve, although the DNA content at the maximum point decreased by 7-9%. At 60 degrees and 1 N HCl, the loss of DNA reaches up to 30%. The prolongation of the hydrolysis time caused even more losses of DNA: at 60 degrees they are equal to 70% (22 minutes following the maximum point), at 37 degrees it is equal to 55% (26 minutes) and at 22 degrees only 9.5% (100 minutes) (the quantity of DNA at the maximum point is taken for 100%). During all the experimental conditions and in both the cell types, the hydrolysis curves are monomeacked, and at 22 degrees starting from 30 minutes a plateau is observed with a slight increase towards the 50th minute. The quantity of DNA-fuchsin in the loose nuclei of monocytes is generally higher than in compact nuclei of erythrocytes. The analysis of scanning- and histograms has shown that the "storage" of DNA in the erythrocyte nuclei and its loss during hydrolysis are related to the fact that apurinization of DNA in the compact chromatin is getting more slowly, whereas its loss due to depolymerization-extraction is higher than in the loose chromatin. These phenomena are expressed least of all during "cool" hydrolysis. The above circumstances should be taken into account during the analysis of nuclei with different degrees of DNA density. Hydrolysis in 5 N HCl at 22 degrees is recommended.