Feulgen Procedure Research Articles

Intranuclear Crystals in the Intestinal Epithelium of the Snail Marisa cornuarietis (Prosobranchia)

Abstract Filamentous intranuclear crystals consisting of two sets of intersecting sheets are common in the intestinal epithelium. They are closely associated with chromatin granules but electron microscope cytochemistry using a modified Feulgen procedure did not show DNA in the crystal itself. Light microscope histochemical tests indicated that the crystals consist of protein.

An Investigation of Feulgen's Procedure for Training Mitotic Divisions in Plants

An Investigation of Feulgen's Procedure for Training Mitotic Divisions in Plants

A Feulgen technique for identification of cucumber chromosomes

A Feulgen procedure was followed for identifying the somatic chromosomes of cucumber (C. sativus L.). The chromosomes were differentiated into dark and light banded regions facilitating the precise karyotyping of somatic complement. Pre-treatment temperature was found to be not critical for producing Feulgen bands in cucumber.

Combined staining procedures for cytophotometry of protein and DNA Feulgen-Naphthol Yellow S and dinitrofluorobenzene-Feulgen.

A comparison has been made between dinitrofluorobenzene (DNFB) and Naphthol Yellow S (NYS) as protein stains in combination with the pararosaniline-SO2 Feulgen procedure. Chicken erythrocytes were used as test cells. Cytophotometric measurements were made using a Zeiss scanning stage cytophotometer coupled to a PDP 11/10 minicomputer using the BICOSCAN program to obtain values for protein per cell, protein per "nuclear area' and DNA per nucleus. With 5N HCl as the Feulgen hydrolysis agent, DNFB staining, applied before the Feulgen procedure, was found to be unaffected by hydrolysis conditions required to give optimum Feulgen staining and showed only small losses after longer hydrolysis times. On the other hand measurements of NYS staining, of necessity applied after the Feulgen procedure, seem to be susceptible to the duration of Feulgen hydrolysis. This susceptibility is probably due to the interaction of the DNA phosphates with the basic amino acid residues, potential binding sites for NYS. Since the degree of this interaction may be variable, it is argued that NYS binding will measure the available basicity of proteins at the time of staining but no specific protein fraction. DNFB binding is unaffected by DNA-protein interactions and therefore can give a more reliable measure of "nuclear' protein, particularly in conjunction with Feulgen-DNA measurements.

MICROFLUOROMETRIC MEASUREMENT OF DNA IN EUDORINA ELEGANS AND E. CALIFORNICA (CHOROPHYCEAE)1

ABSTRACTThe fluorescent dye 2,5‐bis (4′‐aminophenyl (1′))‐1,3,4‐oxidiwle (BAO) was compared to the Feulgen procedure as a method for quantitative comparison of DNA levels in two species of Eudorina. The BAO procedure proved to be specific and as quantitative as the Feulgen procedure, and was more convenient and rapid. The DNA level of the nuclei of gonidial cells of Eudorina elegans Ehrenberg and E. californica (Shaw) Goldstein doubled prior to each division. The DNA level of the somatic, usually non‐dividing nuclei of E. californica did not vary from the IC (haploid) level.

Proliferative and degenerative events in the early development of chick dorsal root ganglia. I. Normal development.

Development of the chick dorsal root ganglia was examined in 4.5- to 9.5-day embryos. Tritiated thymidine (3H-TdR) and autoradiography was used to analyze proliferative activity and the Feulgen procedure to analyze degenerative activity in ganglia 12-17. Proliferative activity was found to be elevated through 4.5 days of incubation when as many as 14% of the ganglionic cells become labelled following a one-hour exposure to 3H-TdR. By 6.5 to 7.5 days proliferative activity decreases to 2-4% in the lateroventral (LV) regions and to approximately 1% in the mediodorsal (MD) regions of the ganglia. However, there appears to be increased proliferative activity by the end of the experimental period at 9.5 days. Birthdate studies demonstrate that large-scale neuronal production occurs between 4.5 and 6.5 days in the LV regions and between 4.5 and 7.5 days in the MD regions. After those times ganglionic proliferative activity must be largely nonneuronal in nature. This nonneuronal proliferation is greater in LV than in MD regions and in brachial than in nonbrachial ganglia. Degenerative activiy was found to be absent from the ganglia until after 4.5 days of incubation. It then increases rapidly, and by 5.5 days 5% of the LV cells in nonbrachial ganglia are degenerating. Degenerative activity then declines but is still present at 9.5 days. In contrast to results of an earlier study (Hamburger and Levi-Montalcini, '49), degenerative activity was also found in the LV region of brachial ganglia and the MD regions of brachial and nonbrachial ganglia. The pattern of LV degenerative activity in brachial ganglia is similar to that in nonbrachial ganglia, but the level of activity is lower. In the MD regions degenerative activity increases throughout the experimental period, and by 9.5 days as many as 4% of the MD cells are degenerating.

Proliferative and degenerative events in the early development of chick dorsal root ganglia. II. Responses to altered peripheral fields.

Responses of chick embryo dorsal root ganglia to early wing bud amputation were examined histologically using tritiated thymidine (3H-TdR) and autoradiography to analyze proliferation and the Feulgen procedure to visualize degenerating cells. Right wing buds were amputated at stage 15 or 16. At 4.5 to 9.5 days of incubation embryos were given a 1-hour exposure to 3H-TdR and fixed. Feulgen-stained autoradiographs were examined for percentage of cells labelled (labelling index) or degenerating (degeneration index) in lateroventral (LV) and mediodorsal (MD) regions of brachial (G14-16) and nonbrachial (G12, 13, 17) ganglia. The earliest response to amputation was a highly significant increase in degeneration indices of LV and MD regions of ipsilateral brachial ganglia at 5.5 days. Significant brachial LV responses were observed throughout the remainder of the experimental period. Two peaks occur in this response: at 5.5 days, corresponding to the peak seen in normal nonbrachial ganglia, and at 8.5 days, having no counterpart in normal development. In brachial MD regions significant degenerative responses occur at most times examined. Significant responses also occur at 7.5 and 8.5 days in MD regions of nonbrachial ganglia. The presence of MD responses in our material indicates that maturation of at least some MD neurons occurs earlier than previously thought. Significant labelling responses occur in brachial LV regions from 7.5 days on. Because other studies (Carr and Simpson, '78a) show that this time is after the end of large-scale neuronal production, this labelling response must be nonneuronal in nature. We conclude that this response is a secondary response to amputation, consequent to the greatly increased cellular degeneration. Results of experiments involving addition of limb buds at the brachial level are also presented.

Effect of Friend leukemia virus on megakaryocytes and platelets in mice.

The thrombocytopenia that follows infection of C3H/Bi mice with Friend leukemia virus (FV) has been investigated. At various times after infection, megakaryocytes in bone marrow and spleen were collected on Nuclepore filters, stained with the Feulgen procedure, and counted. At the same times, platelets in circulating blood were enumerated by the direct method of Brecher et al., 1963 We found that megakaryocyte numbers decreased within 3 days of infection; platelet numbers began to fall at 7 to 9 days. At 11 days megakaryocyte numbers in femoral marrow and spleen were reduced to less than one-third of control values, platelets to about one-quarter of control values. The data are compatible with the hypothesis that the thrombocytopenia which occurs after FV infection in mice results from the reduction in megakaryocytes that is brought about by the infection. Whether all classes of megakaryocytes are equally affected by FV was next examined. At various times following infection, megakaryocytes in marrow suspensions were concentrated by unit gravity sedimentation and microspectrophotometric measurements of nuclear DNA content were made. The kinetic data showed that, following FV infection, megakaryocytes of high nuclear DNA content were markedly reduced in number, but those of low nuclear DNA content were relatively unaffected. Thus the virus infection may interfere with the normal sequential doubling in DNA content of megakaryocytes and/or it may lead to preferential elimination by the host of those megakaryocytes in the higher DNA classes.

Quantitative cytochemical aspects of a combined Feulgen-Naphthol Yellow S staining procedure for the simultaneous determination of nuclear and cytoplasmic proteins and DNA in mammalian cells

The simultaneous cytophotometric determination of nuclear and cytoplasmic proteins and DNA by means of a combined Feulgen-Naphthol Yellow S (NYS) staining procedure was investigated. According to this procedure Feulgen staining is performed prior to NYS staining. The following main results were obtained: 1. 1. After NYS staining alone, the amount of NYS bound to the cell was found to be closely correlated to the cellular dry mass. The correlation coefficient was 0.99 in ethanol-acetone fixed cells and 0.95 in formaldehyde-fixed cells. This close correlation was not significantly altered by the Feulgen staining procedure and was 0.92 in ethanol-acetone and 0.94 in formaldehyde-fixed cells. However, the absolute amount of NYS bound per unit dry mass was affected by the method of fixation and type of Feulgen hydrolysis. 2. 2. The cells lose material during the Feulgen procedure, particularly during the acid hydrolysis stage. The type of hydrolysis most suitable for the Feulgen procedure (5 N HCl, 22 °C, 60 min) resulted in a considerable loss of dry mass in ethanol-acetone fixed cells. This loss was smaller in formaldehyde-fixed cells (15%) and was in addition closely correlated (correlation coefficient 0.99) to the dry mass of the cells prior to hydrolysis. In formaldehyde-fixed cells the dry mass after the Feulgen procedure is thus a good measure of the true cellular dry mass of the fixed cells. This is further demonstrated by the close correlation between NYS binding to Feulgenstained cells and the dry mass of these cells prior to the Feulgen procedure (correlation coefficient 0.95). 3. 3. When using the combined Feulgen-NYS staining procedure under standardized conditions (formaldehyde fixation and acid hydrolysis in 5 N HCl, 22 °C, 60 min) a constant amount of NYS was found to be bound per unit dry weight to nuclear and cytoplasmic proteins in various types of mammalian cells with different proliferative activity. 4. 4. The Feulgen DNA determination was not found to be quantitatively affected by the subsequent NYS staining. From the results of the present study it seems that, under standardized conditions, the combined Feulgen-NYS staining procedure can be used as a reliable quantitative method for the determination of nuclear and cytoplasmic proteins and DNA in mammalian cells.

Microspectrophotometric estimates of non-histone proteins in cell nuclei displaying differing degrees of chromatin condensation.

Nuclei isolated from mouse thymus, kidney, and liver were fixed in ethanol-acetic acetic acid; treated with dilute acid to extract histones; stained by three protein end-group procedures; and measured by scanning, integrating microspectrophotometry. Measurements were also made of nuclei isolated from the same organs and stained by the Feulgen procedure for DNA. Protein end-group procedures included pH 2.8 Biebrich scarlet (for basic groups), mercury orange (for sulfhydryl groups), and mercury orange after thioglycolate reduction (for the sum of sulfhydryl and disulfide groups). With the exception of the comparison between Feulgen-stained 2C liver and kidney nuclei, the integrated extinction values obtained for nuclei of a given organ differed significantly from the measurements of nuclei from other organs, regardless of the staining procedure. Furthermore, the integrated extinction values for 2C nuclei were highest in larger, more vesicular nuclei (from liver and kidney) and lowest in condensed thymocyte nuclei, except in the case of measurements of the disulfide content of the nuclei. In this instance, the values of integrated extinction were highest in condensed thymocyte nuclei, intermediate in kidney nuclei, and lowest in 2C liver nuclei. When 2C, 4C, and 8C liver nuclei were compared, the integrated extinction values of 4C nuclei were found to be approximately twice those of 2C nuclei whose disulfide and Feulgen values were, respectively, higher and lower than expected. The greater disulfide values and reduced Feulgen values obtained in thymocyte and 8C liver nuclei might be related to a greater degree of chromatin condensation in these nuclei, and therefore, to a reduction or selective restriction of their RNA transcriptional capacities.

RNA-Synthese in der Mitose I. Beziehungen zwischen dem Kondensationsgrad der Chromosomen und dem Einbau von 3H-Uridin

Introduction. Depression of RNA synthesis is well documented, but there are only vage suggestions on the regulation mechanism involved. One of the reasons discussed is the condensation of chromatin which could repress the template activity of DNA. The aim of the present study was to look for possible relationships between RNA synthesis and the degree of condensation during mitosis. Materials and Methods. Asynchronous cultures of L-cells were incubated for 10 minutes with 1 μC/ml 3H-uridine and fixed. Mapped mitotic cells were grouped into 12 different stages covering early prophase through early interphase. Following counting of silver grains after autoradiography the cells were stained according to the Feulgen procedure. Two different parameters were used as a measure for the “degree of condensation”: 1. The mean optical density measured with the scanning cytophotometer UMSP I (C. Zeiss) and 2. The reciprocal surface area occupied by the Feulgen stained material. This latter parameter was elaborated by photographing the cells and planimetry of the enlarged images. In addition the optical path difference in the cytoplasm, condensed and uncondensed chromatin of pro- and interphase nuclei and in chromosomes of mid-mitosis cells was measured. A Zeiss Jamin-Lebedeff-type interference microscope was used for this purpose. The relative frequency, i. e. the duration of the different mitotic stages was also counted. Results. The number of silver grains during mid-mitosis (late prophase to late telo-phase) was roughly ten percent of that of early prophase or early interphase (fig. 1). These changes in RNA synthetic activity occur within less than 15 minutes. Optical path differences do not considerably change in the cytoplasm (fig. 2) and in the heteropyknotic part of the chromatin, while in mid-mitosis the isopyknotic chromatin acquires a density comparable to that of the heteropyknotic chromatin (fig. 3). During metaphase reciprocal area and mean optical density rise to values two to three times that of interphase and early prophase (fig. 4 and 5). There exists a significant correlation between the “degree of condensation” of chromatin and the number of silver grains, which is thought to reflect RNA synthesis. This correlation can be demonstrated during the condensation process in pro-phase (fig. 6) as well as during the decondensation of chromosomes in telophase and early interphase (fig. 7). Discussion. The observed correlation between the compactness of chromosomes and their ability to prime for RNA synthesis can be interpreted as a causal relationship. Depression of RNA synthesis may during mitosis merely be the result of chromosome condensation with no specific regulation mechanism involved. Under these aspects the mitotic chromosome is a model for the regulation of transcription processes by chromatin condensation.

Artefactual staining of the peripheral zone of needle biopsies.

A description of artefactual staining in 156 needle biopsies of the liver is given. The following representative methods were employed: Alcian blue'basic fuchsin mixture pH 3, fast green FCF pH 8, azure A'eosin Y mixture pH 5, gallocyanin'chromalum, Feulgen procedure and aluminium'haematein. Changes in stainability were seen in the 3–15 most peripheral cell layers and affected both cell nuclei and cytoplasm as well as sinusoidal lining. A dissociation of nucleoprotein and denaturation of protein in the outermost layers of the biopsy effected by the needle is suggested as being the cause of the altered stain‐ability. The consequences of these findings for the evaluation of needle biopsies is discussed.

An improved flow microfluorometer for rapid measurement of cell fluorescence

An improved instrument for the making of high-speed fluorescent measurements on single cells has been constructed and characterized. Instrumental optimization was accomplished with Chinese hamser cells (line CHO) stained by the fluorescent Feulgen procedure using either auramine-0 or acriflavine. Contributions of instrumental resolution and artifacts to cellular DNA distributions have been determined. The instrument introduces a total coefficient of variation of less than 2% for the case of CHO cells stained with bright dyes such as acriflavine, where photon-statistical effects are minimal. The design of the instrument is such that any of a number of cellular constituents or properties can be studied and quantitated on a single-cell basis at a rate of 50 000 cells/min.

Quantitative zytochemische Untersuchungen der Veränderungen des Nukleoproteins von Spermien bei Fertilitätsstörungen des Mannes

Introduction There is some evidence that fluctuations of the Feulgen-DNA content of spermatozoa associated with male infertility do not represent true differences in DNA content. It is rather the changes of the nuclear proteins modifying the stoichiometry of the Feulgen reaction which seem to be responsible for these facts ( Gledhill et al., 1966 ). These phenomena should be more precisely defined, as proposed by Sandritter et al. (1965) by investigating the kinetics of the “Feulgen hydrolysis curve” of the spermatozoa. Material and Methods Ejaculated spermatozoa were fixed immediately in 96% ethanol, hydrolyzed, as proposed by Bachmann (1968) at 39.5°C in 0.75 N HCL for 2, 3.5 and 5 hours, and stained with Schiff's reagent ( Graumann, 1953 ) according to the Feulgen procedure. The total dye content of the stained spermatozoa was determined using a type GN 2 scanning integrating microdensitometer (Barr & Stroud, Glasgow, Scotland). The nuclei of 50 sperm heads of each slide were measured at 570 nm, the mean value and the variation of the measurements calculated. The significance of this variation between the spermatozoa of fertile and infertile men was checked by an F-test. Furthermore, the hydrolysis curves of spermatozoa of fertiles and infertiles were compared. Fertility and infertility were identified by standard andrological examinations. Results and Discussion The hydrolysis curve of spermatozoa shows a fertility-linked significant difference as can be demonstrated by Figure 1. The mean values of dye content in arbitrary units (AE) of the spermatozoa of fertile male are lower and the curve does not reach a maximum after 5 hours of hydrolysis, indicating a different stoichiometry of the Feulgen reaction. The variations (s 2) of our cytophotometric measurements also show a difference between the populations of fertile and infertiles spermatozoa. The latter show a significant higher scattering of the cytophotometrically determined dye content after different hydrolysis times. The results of these calculations are summarized in Figure 2 and Table 1, insignificant values being drawn with heavy-faced typs. All these findings are considered as fertility-linked disturbances in synthesis and structure of acid nuclear proteins and the so-called (acid-insoluble) “residual proteins” of the sperm heads. Considering data of Bahr and Wied (1971) this control of protein synthesis seems to be lax under physiological conditions with respect to quantity but not to quality. This means that the macromolecular components of sperm cells are assembled without regard to strict proportionality. It now seems obvious that under pathological conditions linked to infertility, which is expressed by the abnormal histological pattern of spermiogenesis (Fig. 3), the lack of control of protein synthesis may increase. This simple investigation of the structure of nucleoproteins of the sperm head enables us to investigate the complex problems of male infertility.

DNA constancy in heteroploidy and the stem line theory of tumors.

Cellular DNA was measured by high-speed flow microfluorometry in mammalian diploid and heteroploid cell populations stained by the fluorescent Feulgen procedure. Heteroploid cells with elevated modal chromosome number showed the expected increase in modal DNA content. However, the variability of DNA content was the same in diploid and heteroploid cell populations despite the large variability of chromosome number in the latter populations. This suggests that heteroploidy may include defects in the chromosomal condensation and kinetochore development systems.

Staining Chromosomes in Sections of Germinal Vesicles of Sarcoptid Mites by a Schiff Reagent

Chromosomes of oocytes, especially early prophase I stages, of Acaridae and Anoetidae species are difficult to stain by procedures using hematoxylin, Feulgen and aceto-orcein. Hematoxylin stains are intensely polychromatic in oocytes; the standard Feulgen procedure is negative with chromosomes during diffuse prophase stages. Satisfactory staining can be obtained with a supersensitive Schiff reagent (Tobie, W. C., Ind. Eng. Chem., Anal. Ed., 14: 405—406, 1942) made by reducing basic fuchsin with gaseous SO2. Routinely prepared paraffin sections of mites fixed in Carnoy's 6:3:1 mixture were hydrolysed 5-8 min in 1 N HCl, washed well, and stained in this reagent: 1-2 hr for prophase oocytes, 10-20 min for condensed chromosomes. A second staining in a 0.5% aqueous solution of toluidine blue 0, adjusted to pH 5.3-5.5 with a citrate buffer, served to darken the original Feulgen stain. Counterstaining with 0.1-0.2% fast green FCF in the last fluid of the dehydrating series enhanced contrast between chromosomes and cytoplasm. This staining technic is also suitable for preparing whole mounts of mites.

A root squash technique for counting somatic chromosomes in sugar cane.

Sugar cane cuttings after treatment with 0.75% Aeratan (organic mercurial fungicide) were germinated in a soil mixture at 26–28 C and transferred to an incubator at 33–34 C for 1 day before collecting roots. The excised roots were pretreated with a saturated solution of α-bromonaphthalene in 0.05% saponin for 3 hr at 8–10 C, fixed in 1:3 acetic-alcohol for 48–72 hr at room temperature, hydrolysed in 1 N HCl for 7–8 min at 60 C, treated with 3% pectinase solution in pH 3.6 acetate buffer for 60–90 min, stained by the Feulgen procedure, and squashed in 1% acetocarmine. Following this procedure, excellent preparations were obtained for counting chromosomes, in which the details of chromosome morphology were often clearly visible. The important precautions for the successful application of this technique include: avoidance of excessive watering of the germinating cuttings, avoidance of sudden temperature changes during processing of unfixed roots, ensuring correct acid temperature during hydrolysis and, final...

Cotton embryogenesis: The identification, as nuclei, of the X-bodies in the degenerated synergid

The "x-bodies" present in the degenerated synergid of cotton were shown to contain DNA by specific staining with Azure B, the Feulgen procedure, and by labelling with (3)H-actinomycin D. They are identified on a morphological basis as the degenerated vegetative nucleus of the pollen tube which is always found in the degenerated synergid tip, and as the degenerated synergid nucleus, which is found about halfway up the degenerated synergid near the side towards the central cell. The positions of these nuclei and of some other structures have been used to construct a tentative description of some of the events occurring during pollen-tube discharge and movement of the sperm through the synergid. Some observations which may indicate some of the factors involved in interaction of the synergid and pollen tube cytoplasm are discussed.

Combined protein and DNA measurements in plant cells using the dinitrofluorobenzene and feulgen techniques.

SYNOPSISThe dinitrofluorobenzene reaction without subsequent chemical treatment has been shown to be suitable for combination with the Feulgen procedure to provide a means of measuring protein and DNA in the same cell. The density of dinitrofluorobenzene colouration per cell is affected by the fixation procedure and by the composition and temperature of the DNFB solution but with all the treatments used, the same reproducible maximum optical density per cell was obtained by increasing the temperature at which the reaction was carried out. The Feulgen procedure has no effect on the optical density of cells previously treated with dinitrofluorobenzene and is not affected itself by this previous staining procedure.

Structural aspects of amitosis: a light and electron microscope study of the isolated macronuclei of Paramecium aurelia and Tetrahymena pyriformis

Thin sections show the macronuclei of Paramecium aurelia and Tetrahymena pyriformis to contain two types of bodies. The smaller, measuring 0.1–0.2 μ in diameter, have been resolved in the light microscope by first removing the macronuclei from the cells in the presence of Mg++, then chelating that divalent cation with EDTA, resulting in expansion of the nuclear material. By staining with methyl green, Azure B, and the Feulgen procedure, the small bodies were shown to contain DNA. In whole mounts these small bodies appear to be joined to one another producing a complex network suspended in which are the larger bodies, or nucleoli. — Macronuclei from both ciliates were isolated in large quantities and purified for spreading on an air-water interface. When the nuclei burst from surface tension forces and are examined with the electron microscope, the DNA containing bodies remain attached to one another by means of 100 A fibrils. The pattern of attachment is non-linear. Occasionally individual DNA-containing bodies “loosen” revealing a coil resembling both in shape and dimensions the 250 A coil characteristic of eucaryotic chromatin. The substructure of the 250 A coil has not been directly observed. However, the frequent association of pairs of 100 A fibrils makes it likely that two such fibrils are tightly complexed in the 250 A coil. The 100 A fibril, in turn, contains two 20 A strands, each presumably a DNA double helix. — In Paramecium each small body of the macronucleus contains approximately one chromosome-equivalent of DNA. The fact that these small bodies are joined to form large structured masses of chromatin within the macronucleus indicates that the distribution of genetic material is not random. It is possible that, similar to bacteria, entire genomes segregate as units, thus accounting for successful amitotic division.