I. Quantitative determination of the amount of DNA per nucleus by interference microscopy

A method for the determination of the DNA content of isolated nuclei of different ploidy has been developed. It is based on measurement of the nuclear dry mass, with an integrating microinterferometer, before and after DNase treatment. The values found are slightly low, because, as indicated by biochemical determinations, consistently 5% to 8% of DNA is not extracted by DNase under these conditions. The average DNA values thus obtained for diploid and tetraploid nuclei of adult rat liver are 7.7 and 15.6 pg (10^-12 g), respectively. Definite advantages of this procedure are: i) comparisons with biochemical determinations to give DNA values for each class of ploidy, ii) comparisons with histophotometry of the Feulgen dye-DNA complex to give absolute values instead of arbitrary units.     

Quantitative cytochemical determination of desoxyribonucleic acid with the Feulgen nucleal reaction

The possibility of using the Feulgen nucleal reaction for a quantitative cytochemical estimation of desoxyribonucleic acid (DNA) was investigated. The intensity of the reaction in nuclei was determined by absorption measurements with the microscope. The accuracy of such measurements was tested by comparison with measurements on the same material with a Beckman spectrophotometer. The values obtained with the microscope agreed within a few per cent with those obtained with the Beckman spectrophotometer. Furthermore, the errors introduced by uneven distribution of absorbing material, by variations in the numerical aperture of the system, and by variation in the area used on the phototube were investigated empirically. The following variables were studied with regard to their effect on the intensity of the Feulgen reaction: type of fixation, time of hydrolysis after acetic acid-alcohol and formalin fixation, time of staining in leucobasic fuchsin, method of preparation of leucobasic fuchsin. The intensity of the Feulgen reaction in liver and erythrocyte nuclei of various vertebrates, fixed in acetic acid-alcohol, was then compared with the DNA content of these nuclei as determined by chemical analysis on a known number of nuclei. The intensity of the reaction was found to be proportional to the DNA content of the nuclei, if nuclei of similar structure and DNA concentration were compared. In nuclei of different structure and DNA concentration (i.e. liver and erythrocyte nuclei), fixed in acetic acid-alcohol, the intensity of the Feulgen reaction was, however, not proportional to the DNA content. This difficulty was overcome by isolating nuclei in sucrose and by fixing them in formalin. Uniform distribution of DNA and therefore uniform coloring after the Feulgen reaction were thus obtained. In such nuclei with uniform distribution of absorbing material the Feulgen reaction was found to be proportional to the DNA content of nuclei, even if they differed greatly in their DNA concentration. The Feulgen nucleal reaction is not quantitative in an absolute sense. For absolute determinations nuclei of known DNA content must be treated together with the unknown material to serve as standard. From these data it therefore appears possible to determine cytochemically relative amounts of DNA in cellular structures by measuring their absorption after treatment with the Feulgen nucleal reaction.     

A study of DNA depolymerisation during Feulgen acid hydrolysis.

The binding of Schiff dye molecules after acid hydrolysis (1 M HCl) for varying lengths of time was studied in ascites tumour cells. The amount of dye bound to the tumour cells closely followed the number of aldehyde groups, calculated from the extraction of radioactive nucleotides. This constant dye to aldehyde ratio did not change when the hydrolysis was performed at a lower acid concentration (0.3 M HCl). The conclusion drawn is that Feulgen dye measurements represent, in a constant way, the number of aldehydes on DNA at any given time during hydrolysis. The alteration of the hydrolysis pattern of chromatin fixed in formalin was found to be due to a slower extraction of DNA depolymerisation products, the purine liberation being unaffected. A similar explanation is offered for the extreme pattern obtained from hydrolysis of bull spermatozoa chromatin.     

Standardization of the Feulgen reaction for absorption DNA image cytometry: a review.

The present paper gives a review of the current potentials and problems of a standardized Feulgen reaction for absorption DNA image cytometry. The cytochemical basis of the Feulgen reaction is described in the first part of this review. Subsequently, several preparatory factors which influence the performance of the Feulgen reaction, such as fixation, acid hydrolysis, composition of the Feulgen reagent and, in histology, embedding, are discussed in more detail. Some user-oriented recommendations for a standard Feulgen technique are given.     

Absorption cytophotometry: evaluation of three methods for the determination of DNA in Feulgen stained nuclei.

Three methods for the evaluation of the relative amount of DNA per nucleus by absorption cytophotometry are compared. A combination of the two-wavelength method (Patau, 1952; Ornstein, 1952), the one-wavelength two-area method (Garcia, 1965) and the determination of transmission through nuclear plugs is proposed in order to estimate systematic errors made by absorption cytophotometry.     

Factors affecting the course of DNA acid hydrolysis in carrying out the Feulgen reaction]

Literature data concerning acid hydrolysis of DNA during the Feulgen procedure are reviewed, with emphasis being made on the dependence of Schiff-apurinic acid binding on the fixation technique, the temperature of hydrolysis and acid concentration, the rate of extraction of depolymerized DNA fragments, the nucleotide composition of DNA, the chromatin state, and on the composition of nucleoprotein. Some practical considerations for optimization of the Feulgen procedure for a precise quantitative determination of DNA amount are given.     

Some aspects of the Feulgen reaction in situ

Summary Cytological observations combined with studies on absorption spectra of Feulgen stained normal and lipid — extractet HeLa and ehrlich-Lettré mouse ascites cells were performed after fixation of the cells as well in neutral formaldehyde as in Serra fixative. The effects of formaldehyde treatment of the stained cells to substitute all the free amino groups of DNA bond pararosaniline molecules, were also studied. The results obtained by using DNA samples containing 2% protein and relatively free from protein, led to the conclusion that after acid hydrolysis for a short period purines in DNA become splitted and these released aldehydes react with one or two amino groups of pararosaniline, a triphenylmethane dye (according to the arrangement of purines and pyrimidines in the helices). Some protein molecules also take part in the reaction and substitute some of the free amino groups of DNA bound pararosaniline. Peulgen stained cells fixed in Serra fixative show an absorption maximum at 546–550 m. Under appropriate conditions, as in cells fixed in formaldehyde, other substances e.g. phospholipids and lipoproteins interfere with the reaction by substituting most of the free amino groups of DNA bound pararosaniline molecules. It has been argued that in histochemical reactions monosubstituted pararosaniline molecules should be coloured and further substitution of free amino groups of pararosaniline, bound in DNA helices, does not change the intensity of the colour, but gives a shift in the wavelength of the absorption spectra.It has been suggested that the differential response of the nucleoli to the Feulgen-reaction, depending on whether the cells were fixed in formaldehyde or in Serra fixative, may be due to the formation of a protecting shield around the finely distributed intranucleolar chromatin strands, when formaldehyde is being used. After this fixation lipoproteins and other lipids, present in a relatively high percentage and closely associated with the intranucleolar chromatin strands, are especially well preserved.Evidences have been put foreward in support of the amino alkylsulfonic acid theory of Rumpf (1935) and Hörmann et al. (1958) whereas the amino sulfinic acid theory to explain the Schiffs reaction (Wieland and Scheuing, 1921) was shown not to be in agreement with our results.On leave from the Department of Botany, Calcutta University, 35, Ballygunge Circular Road, Calcutta-19, India; on a fellowship from the German Academic Exchange Service.     

Repetitive DNA and Feulgen hydrolysis kinetics in three species of Bufo.

Two North American species of the genus Bufo (Bufo cognatus and Bufo boreas, 2n = 22) and one African species (Bufo regularis, 2n = 20) were analyzed with respect to their repetitive DNA fractions and the behaviour of their chromatin to the acid hydrolysis at different times. The mean melting point of the total isolated DNA decreased from 89 degrees C to 87 degrees C with a genome size increase from 4.4 to 7.5 pg. The differences in genome size can only partly be explained on the basis of repetitive DNA fractions (renaturing up to Cot 10 in 0.12 M phosphate buffer). Several fractions in this repetitive range behave independently in the three species and the spectrum of repetitive fractions in the African Bufo regularis differs distinctly from those of the American toads. When fixed chromatin of these species in histochemical preparations is hydrolyzed with 5N HCl during the Feulgen reaction, the kinetics of depurination are equal in all species, while hydrolytic DNA breakdown proceeds distinctly more slowly in Bufo reularis as compared to the other species.     

Feulgen nucleal reaction. I. Quantitative extraction of fuchsin from Feulgen-stained nucleoprotein

A new extraction method for the quantitative determination of the fuchsin contained in a Feulgen-stained nucleoprotein sample has been introduced. The method is based on the following facts: (1) Treatment of a Feulgen-stained nucleoprotein sample with hot acid or alkali brings about a splitting of the linkage between fuchsin-SO(2) and the hydrolyzed nucleic acid moiety of nucleoprotein through aldehyde groups. (2) It also effectuates the formation of fuchsin from the liberated fuchsin-SO(2). (3) The fuchsin is made colorless by the treatment, but is restored to its original pink colored state when the pH of the acidic or alkaline medium is adjusted to 4.6. (4) The fuchsin, either pink colored or decolorized by alkali, can be extracted from an aqueous phase by amyl alcohol. A linear relationship was found to exist between the amount of fuchsin extracted by the FEM from a Feulgen-stained nucleoprotein sample and its DNA content. This relationship holds over a wide range of DNA concentration. From experiments utilizing this method, knowledge may be gained about the mechanism of the Feulgen reaction in situ which can lead to an improvement of the reaction in the field of cytochemistry.