Computer analysis of Feulgen hydrolysis kinetics.

A least squares fit of Feulgen hydrolysis time curves to the Bateman function is performed using an especially adapted parameter transformation together with a standard conjugate gradients iteration procedure. The method has been applied to a large number of measured data, and the use and limits of the computer evaluation are discussed.     

Computer analysis of Feulgen hydrolysis kinetics

Summary A least squares fit of Feulgen hydrolysis time curves to the Bateman function is performed using an especially adapted parameter transformation together with a standard conjugate gradients iteration procedure. The method has been applied to a large number of measured data, and the use and limits of the computer evaluation are discussed.     

A model of the breakdown and removal of the chromatin components during Feulgen acid hydrolysis

Synopsis A stochastic model of Feulgen hydrolysis is proposed. The model, constructed so as to embody the main features of chromatin structure, is simple enough to allow the calculation of extraction rates. Extraction rate curves generated by the model are compared with experimental curves obtained under various conditions (different fixatives and hydrolysis solutions). A good correspondence is found between the experiments and the predictions of the model.     

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.     

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.     

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.     

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.     

Exposure and removal of stainable groups during Feulgen acid hydrolysis of fixed chromatin at different temperatures

Summary Hyperdiploid Ehrlioh's ascites tumour cells grown in male mice (strain NMRI) were labeled with radioactive nucleotides. The nucleic acids were extracted from fixed, air-dried smears by fractionated hydrolysis and their radioactivity measured by liquid scintillation. The experiments showed that the exposure of aldehydes through removal of purine bases and the elimination of these aldehydes through depolymerisation of DNA were the two main processes responsible for the Feulgen hydrolysis curve. They were shown to be independent and overlapping. The depurination can be described as a simple hydrolytic reaction, while the extraction of DNA depends on a number of different factors. This entails that, in the Feulgen acid hydrolysis procedure, the part of DNA measured is dependent upon the stability of the chromatin. It was found that it is possible accurately to determine the depolymerisation process and thereby roughly correct the measured amount of Feulgen DNA.     

Temperature and acid concentration in the search for optimum Feulgen hydrolysis conditions.

Exposure and removal of aldehyde groups during Feulgen acid hydrolysis were studied at a wide range of temperature and acid concentrations. Temperatures between 9 and 75degreesC were found to influence only the rate of the hydrolysis reaction over the entire range from high (6 M) to low (0.05 M) HCl concentrations. The temperature dependence was high, and around +5degreesC was sufficient to double the reaction rate. The influence of acid concentrations between 0.02 and 6 M was studied, and the extraction rates that determine the peak values of the Feulgen hydrolysis curve were found to depend in the same way on the (H+) concentration. A diagram is given that makes it possible to determine the time to reach the point during hydrolysis where the maximum amount of aldehyde groups are developed for a wide range of temperatures and acid concentrations. Temperatures slightly above room temperature in combination with high acid concentration is recommended for Feulgen hydrolysis.     

Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology

Sperm chromatin alteration is an important feature that can affect fertility of the male rabbit. This study compared toluidine blue staining with Feulgen reaction (as methods for evaluating chromatin alteration) and investigated the relationship between sperm morphology and chromatin alteration. Seven hundred rabbit ejaculates of animals with unknown fertility were used. Primary and secondary morphological sperm abnormalities were evaluated in semen smears with phase-contrast microscopy. Chromatin alterations were evaluated in semen smears stained with toluidine blue (pH 4.0 and 5.0) and with the Feulgen reaction. While the three methods were equally efficacious for identification of chromatin alterations, toluidine blue staining was more appropriate to characterize the intensity of chromatin alterations. The correlation between primary sperm defects and chromatin alteration was high and positive, suggesting that sperm chromatin structure affected sperm head morphology. The correlation between secondary sperm defects and chromatin alteration was also positive, but lower. The final chromatin compaction occurs in the epididymus, where secondary sperm defects originate. Therefore, the causes of secondary sperm defects could also intervene with final chromatin compaction. In summary, the toluidine blue stain was an effective means of evaluating the sperm chromatin alteration in rabbit spermatozoa.     

Studies on the kinetics of reaction and hydrolysis of fluorescamine

The influence of various parameters on the rate of reaction of fluorescamine with primary amines and on the rate of hydrolysis of the reagent is described. The studies indicate that both are dependent on the reaction conditions, including pH, solvent in which the reagent is prepared, temperature, reagent concentration, and buffer salt. Under any set of conditions the reaction rates vary with the amines. A correlation between reaction rate and extent of fluorophor formation has been demonstrated. Kinetic evidence for a multistep reaction mechanism, as well as values for the kinetic constants, are presented.     

The effect of sodium chloride on the extraction of DNA fragments during Feulgen acid hydrolysis

Synopsis Feulgen acid hydrolysis was performed on ascites tumour cells labelled with radioactive DNA-precursors. The development of fragments of apurinic acid and the extraction of purines were studied by monitoring the variations in the extraction rate during the hydrolysis when sodium chloride was either present or absent from the hydrolysis solution. The changes in the rate of extraction of purines and the alterations in the initial retardation of the apurinic acid extracting process followed approximately the same pattern. The extractability of apurinic acid fragments during hydrolysis in 0.3m HCl was found to be a maximum when the sodium chloride concentration was about 1m. Sudden exchange experiments, in which acid was substituted for sodium chloride after various times of hydrolysis, revealed a successive shortening of the extractable fragments during the low acid concentration hydrolysis. The results strengthen the view that, during hydrolysis, apurinic acid is lost from the cells through a reaction whose form is determined, first, by an initial retardation of the depolymerization, second, by the maximum length at which fragments developed through the depolymerization become soluble and are lost by diffusion, and last, at low acid concentrations, by a mechanism whose influence is equivalent to the presence of bonds between the fragments and an unextractable stable structure.     

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.