Absorption-Spectrophotometric Determination of DNA (Deoxyribonucleic Acid) in Milk Ad Modum Feulgen

The Feulgen reaction is examined by absorption photometric measurements at 565 nm with correction for varying background absorption at 485 nm. This correction is done to improve the accuracy of measuring. Examinations of the accuracy of analysis for the Feulgen reaction and the direct microscopic cell counts show that the former is of the same order, when the cell count is made at a working factor of 17,000. The standard deviations expressed in percentage of the reaction value are greater the lower the reaction is, whereas the absolute standard deviation is reduced. It has been demonstrated that addition of a few (< 5) µg of DNA per ml milk can give Feulgen reaction. The coefficient of correlation between log added amount of DNA and log Feulgen reaction is 0.98. The coefficient of correlation between log Feulgen reaction and log cell content depends on the accuracy at which both determinations are made. In a determination of cell content at a working factor of 550, and several determinations of the Feulgen reaction the coefficient of correlation (r) is found to be 0.98. A cell content determination at a working factor of 20,000 and a single determination of Feulgen reaction on each milk sample yields r = 0.83. An examination of foremilk samples of the same cell content reveals on an average the highest reaction in mastitis-affected quarters which have been infected within the last 4 weeks. Quarters that have been infected for more than 4 weeks, on an average show the second highest reaction, whereas quarters of physiological cell number show the lowest reaction. The DNA-content in most cases is found to be too high compared with the number of cells counted microscopically. The DNA-content in centrifugated cells corresponds to the one calculated theoretically. The surplus DNA-content can be demonstrated in the cell-free skim milk fraction, probably originating from destroyed cells. The studies performed suggest that determination of the content of 2-deoxyribose in milk by means of the Feulgen reaction is a more correct measure of the cell content in the milk than is the microscopic cell count. Studies are being continued for illustration of these conditions.     

Changes in the ultrastructure and DNA and protein content in human atrial myocytes in cardiac hyperfunction due to mitral valve defects

Biopsies from human right auricles were obtained during open heart surgery, prior to valve replacement, from six patients (aged from 20 to 49 years) with rheumatic heart disease. DNA and the total protein contents were measured in isolated myocytes by means of the two wave-length scanning cytophotometry after the double Feulgen and Naphthol yellow S staining procedure. In all the biopsies polyploid hypertrophied myocytes predominate. The hypertrophic, nondegenerated cells and the cells with degenerative changes of varying severity (in the first place, changes of contractile apparatus and membranes) are present. The highest degree of cell ploidy occurs in patients of functional class IV according to the New York Heart Association classification, 72 to 98% of cells displaying octaploid and higher DNA values. With the increase in ploidy of myocytes in series 2c----4c----8c----16c----32c----64c the protein content increases only as 2.0----3.0----5.8----7.8----13.0----16.8. Neither direct correlation between the ploidy level and the degree of cell degeneration, no inverse correlation between the degree of degeneration and the value of ejection fraction was observed.     

The Feulgen reaction 75 years on

 The Feulgen reaction proposed by Feulgen and Rossenbeck 75 years ago is one of the cytohistochemical reactions most widely used in biology and medicine. It allows DNA in situ to be specifically stained based on the reaction of Schiff or Schiff-like reagents with aldehyde groups engendered in the deoxyribose molecules by HCl hydrolysis. The staining intensity is proportional to the DNA concentration. Current applications of the Feulgen reaction are mainly concerned with DNA quantification in cell nuclei by image cytometry for ploidy evaluation in tumor pathology. From the morphological point of view, specific demonstration of DNA in cell structures at the light microscopic level is very little used nowadays. On the other hand, application of the Feulgen principles to electron microscopy have recently allowed specific DNA-staining procedures to be developed for the study of the structural organization of DNA in situ. Accepted: 13 January 1999     

The phase contrast microscope in plant cytology

Phase contrast microscopy affords a rapid and easy way of counting chromosomes from root-tip cells of Angiosperms. The staining method is a modified Feulgen process, the chromosomes being understained before examination under phase. This cuts down even further the time for obtaining satisfactory chromosome counts. Under phase and using a green filter the understained chromosomes in squashes, after only 30 minutes in Feulgen, appear jet black within an almost transparent cell. There is no interference from the cellulose cell walls.     

A combined stain for identifying epithelial cells of the gastric mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: paradoxical concanavalin A staining (PCS) to identify mucous neck cells, periodic acid Schiff-concanavalin A staining to distinguish mucous neck cells from surface mucous cells, and a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: Feulgen hydrolysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; Feulgen hydrolysis-PAS-concanavalin A-Bowie staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

A Combined Stain for Identifying Epithelial Cells of the Gastric Mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: A) paradoxical concanavalin A staining (PCS) to identify mucous neck cells, B) periodic acid Schiff-concana-valin A staining to distinguish mucous neck cells from surface mucous cells, and C) a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: 1) Feulgen hydroIysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; 2) Feulgen hydrolysis-PAS-concanavalin A-Bowic staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

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.     

Feulgen, iron-propionocarmine and cupra-ammonium in preparing algal chromosomes for light microscopy.

A method for obtaining algal chromosomal preparations is described employing the Feulgen method for DNA staining, Fe-propionocarmine as an enhancing stain, and cupra-ammonium to remove cell wall material. Fe-propionocarmine applied as a gradient to the side provides cells stained with the Feulgen stain alone or with the Feulgen Fe-propionocarmine stain, thereby facilitating useful comparison. Where dilute the Fe-propionocarmine enhances nuclear staining without staining orthe organelles; where more concentration it also stains the nucleolus, spindle, spindle polar bodies, pyrenoid and protoplast. Treatment with cupra-ammonium, to remove polysaccharide wall material, followed by neutralization with propionocarmine, enables thinner squashes and better chromosome spreads without loss of differential staining. Preparations mounted in euparal are long-lasting.     

Feulgen, Iron-Propionocarmine and Cupra-Ammonium in Preparing Algal Chromosomes for Light Microscopy

A method for obtaining algal chromosomal preparations is described employing the Feulgen method for DNA staining, Fe-propionocarmine as an enhancing stain, and cupra-ammonium to remove cell wall material. Fe-propionocarmine applied as a gradient to the slide provides cells stained with the Feulgen stain alum or with the Feulgen Fe-propionocarmine stain, thereby facilitating useful comparison.

Where dilute the Fc-propionocarmine enhances nuclear staining without staining other organelles; where more concentrated it also stains the nucleolus, spindle, spindle polar bodies, pyrenoid and protoplast. Treatment with cupra-ammonium, to remove polysaccharide wall material, followed by neutralization with propionocarmine, enables thinner squashes and better chromosome spreads without IOU of differential staining. Preparations mounted in euparal are long-lasting.     

Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry.

Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method). An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescente in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value. The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.     

Diskrepanzen von Feulgen-Wert und DNS-Gehalt

Zusammenfassung Anhand eigener Untersuchungsergebnisse und entsprechender Befunde aus der Literatur wird auf die Fehlermöglichkeiten bei der quantitativen Interpretation von zytophotometrischen Feulgen-Werten hingewiesen. Durch chemische und biophysikalische Veränderungen des Chromatins im Verlauf von Zellreifungen und Zellfunktionsänderungen können trotz gleichbleibenden DNS-Gehalts verschiedene Feulgen-Werte gefunden werden.

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Discrepancies between cytophotometric Feulgen-value and DNA content

Summary Many results of Feulgen cytophotometry published by numerous authors and some new data of our own demonstrate the possibility of pitfalls when interpreting Feulgen values in relation to DNA content of cell nuclei. As a consequence of chemical and biophysical alterations of chromatin in the course of cellular development and during different functional states there are different Feulgen values to be found in cell nuclei containing the same amount of DNA.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation

Four fuchsin analogues (Pararosaniline, Rosaniline. Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches. Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 microns. Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.     

Feulgen DNA stainability of bone tumors after demineralization

Microspectrophotometric DNA analysis of archival bone tumor tissue is often impeded by previous acid demineralization, which destroys Feulgen DNA stainability. To find an alternative to acid for prospective DNA studies of bone tumors in tissue sections, Feulgen stainability of fresh osteosarcoma specimens after demineralization in neutral EDTA was investigated. The reliability of DNA analysis of weakly Feulgen-stained sections from archival tissue was also studied. Demineralization of four fresh specimens in EDTA slightly reduced Feulgen DNA stainability compared to nondemineralized preparations but did not affect the determination of ploidy level. Hydrolysis tests of one diploid and one hyperploid osteosarcoma showed that the staining relationship between control and tumor cells was not altered by EDTA pretreatment. For DNA studies of bone tumors requiring demineralization, EDTA offers a means of retaining nuclear Feulgen stainability. In 22 archival osteosarcoma specimens of varying Feulgen stainability, three different upper limits of light transmission (75, 85, and 95%) were applied to test the significance of background disturbances in relation to nuclear stain intensity. The relationship between the median total extinction of the control and tumor cell populations was not significantly affected by altering the upper transmission limit except in four poorly stained lesions. The control cells of these four specimens exhibited a median total extinction less than one-third of the maximum encountered. The results suggest that weakly stained archival specimens can be tested for selecting those appropriate for ploidy determination.     

Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry

Summary Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method).An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescence in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value.The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.Partly supported by Alexander von Humboldt-Stiftung     

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.     

Quantification of nuclear DNA and intracellular glycogen in a single cell by fluorescent double-staining.

It was found that intracellular glycogen is stabilized against acid treatment when it is stored under dry conditions for three months after methanol fixation. This stabilization allowed quantitative double fluorescence staining for nuclear DNA and intracellular glycogen, in a single cell. A Feulgen nucleal reaction, with acriflavine-Schiff's reagent following 5 N HCl hydrolysis at 25 degrees C for 4 min, was followed by a pararosanilin-Schiff PAS reaction for glycogen. This short term hydrolysis was found to be sufficient for the performance of a acriflavine-Schiff's Feulgen nucleal reaction and to provide good preservation of intracellular glycogen. Quantification of nuclear DNA and intracellular glycogen were consecutively carried out with a digital microfluorometer on a single ascites cancer cell of the AH-13 line stained by this method. It was found that there is a positive linear correlation between the amount of DNA and glycogen in this cell line.     

云南油杉受精过程中新细胞质及蛋白泡的动态观察

云南油杉(Keteleeria evelyniana Mast)在受精前,精核与卵核周围的细胞质鞘不明显。受精后,合子核周围出现细密的新细胞质。应用孚尔根核染色法,可以较清晰地将新细胞质染出,呈现较弱的正反应,而合子的核质及受精前的精核与卵核染色极弱。卵细胞质及其中的蛋白泡均为负反应。原胚形成后,除上层外,其余几层细胞质内开始积累淀粉粒。此时胚原细胞核的孚尔根染色深度有所增加。幼胚形成后,在顶端的胚原细胞群中核的孚尔根染色反应已恢复正常。在原胚及幼胚胚原细胞质中也呈现很弱的正反应。在电镜下,胚原层细胞质及新细胞质中均含有核样电子致密小体或称作染色质小体,而原胚莲座层细胞质及四周套细胞质中的线粒体则不含这种核样小体。因此,大蛋白泡在卵核形成的早期数量不多,当合子形成时含量最高,而随着游离核的分裂进程,蛋白泡以及原卵质均逐渐地解体,在原胚形成后全部消解。     

Observations on the Neocytoplasm and Proteid Vacuoles During the Fertilization of Keteleeria evelyniana

Distributions and dynamics of the neocytoplasm and proteid vacuoles during the fertilization of Keteleeria evelyniana were studied by histochemical methods. Before fertilization cytoplasmic sheath surrounding the male and female gametes was indistinct. After fertilization, the dense neocytoplasm appeared around the zygote. Part of the neocytoplasm is invaded by mitochondria of maternal origin which had collected in large numbers in the perinuclear zone. The mitochondria contain electron compact little body which looks like a nucleus in the cytoplasm, but not observed in the rosette tier cell of proembryo and jacket cells. Hence, it was showed that the neocytoplasm participated in the development of embryo by all these observations. By using Feulgen reaction, the staining reaction of neocytoplasm was positive, the egg nucleus or zygote nucleus was weaker in positive reaction, while the proteid vacuoles were negative. When the proembryo developed, there were a few starch grains accumulated in the other three tiers except the upper tier. The Feulgen reaction was in- creased in intensity in the suspensor tier and embryonal cell tier nuclei. When the young embryo developed, Feulgen reaction became normal in the nuclei of the embryo initials. The embryo initials and Suspensor cells showed very weak Feulgen positive reaetion in the proembryo and young embryo. The development of the large proteid vacuoles was from plastid. During the early stage of egg nucleus, contents of large proteid vacuoles were less. When the zygote was formed, they reached the highest. However, after the zygote produced, the proteid vacuoles and egg cytoplasm were getting disintegrated following the course of fission of free nuclei. After the proembryo formed, the proteid vacuoles were wholly disintegrated.     

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.     

Simultaneous quantification of nucleoproteins and comparison of methyl green-pyronin Y and Feulgen staining in sections of oral squamous cell carcinoma,dysplastic lesions and normal mucosa

A fundamental difference between normal cells and tumor cells is the proliferative activity of the nucleus and nucleolus, which increases progressively from normal to oral dysplastic mucosa to oral squamous cell carcinoma (OSCC). This activity is evaluated routinely using hematoxylin and eosin (H & E) staining, but in some cases, inter-observer variability occurs among pathologists. We evaluated cellular proliferation by staining sections with the methyl green-pyronin Y procedure and the Feulgen reaction. We also compared the efficacy of methyl green-pyronin Y and Feulgen staining for studying nuclear and nucleolar features in oral dysplastic mucosa and in different grades of OSCC. Sections cut from formalin fixed, paraffin embedded blocks of five normal mucosa, 15 dysplastic mucosa, 10 well-differentiated OSCC, 10 moderately differentiated OSCC and five poorly differentiated OSCC cases were stained with Hematoxylin and Eosin, methyl green-pyronin Y and the Feulgen reaction. The mean diameters of the nuclei and number of nucleoli showed significant differences. A progressive increase in diameter of the nucleus and number of nucleoli was observed from normal mucosa through poorly differentiated OSCC. We observed that methyl green-pyronin Y stain is more useful than Feulgen and hematoxylin and eosin for simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine examination.