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.     

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     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size,Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

Summary A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 x oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r=0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r=0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r=0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30–100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm× 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

Genome Size Determination in Peronosporales (Oomycota) by Feulgen Image Analysis

Genome size was determined, by nuclear Feulgen staining and image analysis, in 46 accessions of 31 species of Peronosporales (Oomycota), including important plant pathogens such asBremia lactucae, Plasmopara viticola, Pseudoperonospora cubensis,andPseudoperonospora humuli.The 1C DNA contents ranged from 0.046 (45.6 Mb) to 0.163 pg (159.9 Mb). This is 0.041- to 0.144-fold that ofGlycine max(soybean, 1C = 1.134 pg), which was used as an internal standard for genome size determination. The linearity of Feulgen absorbance photometry method over this range was demonstrated by calibration ofAspergillusspecies (1C = 31–38 Mb) againstGlycine,which revealed differences of less than 6% compared to the published CHEF data. The low coefficients of variation (usually between 5 and 10%), repeatability of the results, and compatibility with CHEF data prove the resolution power of Feulgen image analysis. The applicability and limitations of Feulgen photometry are discussed in relation to other methods of genome size determination (CHEF gel electrophoresis, reassociation kinetics, genomic reconstruction) that have been previously applied to Oomycota.     

Genome size variation inCajanus cajan (Fabaceae): A reconsideration

A recent investigation of genome size in certain samples of the pigeonpea,Cajanus cajan, indicates values from 1.55 pg to 1.99 pg (1C level), which is 1.29-fold variation between accessions. In the present analysis those of these accessions which had particularly high or low DNA contents in that study were subjected to a reanalysis using propidium iodide and DAPI flow cytometry and Feulgen densitometry. Only minor differences in genome size, not more than 1.047-fold, were found with flow cytometry, and no significant differences were obtained with Feulgen densitometry. The previously reported genome size cannot be confirmed. It is about half as large and was determined in the present study as 0.825 pg (1C, propidium iodide flow cytometry,Glycine max as standard) and 0.853 pg (1C, Feulgen densitometry,Allium cepa andPisum sativum as standards), respectively.     

Comparative microphotometric studies of DNA and arginine in plant nuclei

Summary Photometric measurements have been made of the amounts of stain formed in the Feulgen (DNA) and Sakaguchi (arginine) reactions in plant nuclei of differing ploidy.In nuclei of diploid and tetraploid plants of Tradescantia ohioensis and of diploid, triploid and tetraploid plants of Ranunculus ficaria, both Feulgen and Sakaguchi values gave ratios which agreed closely with the ratios of the number of chromatids known to be present. The Feulgen/ Sakaguchi ratio for each of the different types of nuclei measured was very similar both within and between these two species.In the interphase nuclei of five different species, both Feulgen and Sakaguchi values gave bimodal distributions. In the nuclei of differentiating cells, the proportions of values falling into each of the 2C, 4C or 8C classes were the same for both stains.Measurements of the amounts of both stains were made in sequence on the same individual nuclei and a positive correlation found between the two sets of values.In nuclei from differentiating cells of Vicia faba primary roots, the Feulgen/Sakaguchi ratio decreased with increasing distance from the apex.The following suggestions were made from the results: (a) that there is some degree of quantitative constancy of nuclear arginine which parallels that of DNA; (b) that the amount of nuclear arginine, like that of DNA, is doubled during synthesis in interphase; (c) that the syntheses of DNA and arginine in interphase, if not simultaneous, at least occur within the same relatively short period; (d) that there may be a difference in the DNA/arginine ratio between the nuclei of meristematic and differentiating cells.     

Optimization of the histochemical demonstration of DNA using 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B

Summary Previous methods for the histochemical demonstration of DNA were optimized. p-Toluene sulfonic acid as catalyst for hydrazone formation between the aldehydes generated after Feulgen hydrolysis and 3-hydroxy-2-naphthoic acid hydrazide (NAH) was used instead of acetic acid. Modifications of the conditions of the coupling reaction with Fast Blue B reduced the background staining. The optimized histochemical staining method for DNA (NAH-FB-DNA staining) can be performed easily and reproducibly. Without prior Feulgen hydrolysis the optimized method can also be used for the histochemical demonstration of reactive carbonyls undissolved under the given histochemical conditions.Dedicated to Prof. Dr. E. Schauenstein on the occasion of his 70th birthday     

Proportionalitätsfehler bei der Feulgen-Hydrolyse

Zusammenfassung Beim Vergleich des Feulgen-Farbgehaltes verschiedener Zellkerne (Leberzellen, Lymphozyten, Granulozyten und Spermien) traten nach Alkoholfixierung Abweichungen der gemessenen Feulgen-Werte von den nach dem Gesetz von der DNS- Konstanz zu erwartenden DNS-Gehalt dieser Kerne auf. Verglichen mit den Feulgen-Werten der diploiden Leberzellkerne ergaben Lympho- und Granulozyten bei allen Hydrolysezeiten zu niedrige (bis zu minus 20%), haploide Spermien im postmaximalen Hydrolysebereich zu hohe Feulgen-Werte (z. T. sogar höhere Werte als die diploiden Zellkerne). Innerhalb desselben Zelltypes wurden dagegen, auch beim Vergleich der verschiedenen Ploidiestufen der Leberkerne, keine Differenzen festgestellt.Da die an Leukozyten und Spermien beobachteten Abweichungen nach Methanol-Formalin-Eisessig-Fixierung nicht mehr auftraten und auch durch UV-absorptionscytophotometrische Messungen nicht bestätigt werden konnten, muß man annehmen, daß es sich um Proportionalitätsfehler handelt, die auf Hydrolyseunterschieden beruhen.Für die quantitative Feulgen-Cytophotometrie scheint daher die Methanol-Formalin-Eisessig-Fixierung besser geeignet zu sein als die Alkoholfixierung, bei deren Verwendung es leicht zu Proportionalitätsfehlern während der Feulgen-Hydrolyse kommen kann.

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Proportionality errors during feulgen hydrolysis

Summary Comparing the Feulgen dye-content of different nuclei (liver cells, lymphocytes, granulocytes and sperms) after alcohol-fixation deviations were found between the measured Feulgen values and the DNA-content to be expected from the DNA-constancy law. The Feulgen values of lymphocytes and granulocytes proved to be lower (up to minus 20%) than those of diploid liver nuclei at all hydrolysis times, while in the postmaximal range of hydrolysis the values of the haploid sperms were too high (even higher than those of the diploid nuclei). Such differences did not appear when nuclei of the same cell type in different DNA- ploidy classes (liver nuclei) were compared.Those deviations of leucocytes and sperms were no longer found after fixation in methanol-formalin-glacial acetic acid and, in addition, could not be confirmed by UV-absorption measurements. For that reason we suppose them to be due to proportionality errors caused by variations in the hydrolytic behaviour of the different nucleoproteins.Thus fixation in methanol-formalin-glacial acetic acid seems to be more suitable for quantitative Feulgen measurements than alcohol-fixation, which may easily give rise to proportionality errors during Feulgen hydrolysis.Moreover, to avoid any false interpretation of Feulgen data we should suggest controll measurements using another independent method (f. e. UV-absorption), or — if this is impossible — to check the Feulgen values after different fixations and variant hydrolysis times (premaximal, maximal, postmaximal).

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

The Feulgen banded karyotype of the mouse: analysis of the mechanisms of banding

The chromosomes of the mouse have been identified by specific banding patterns revealed by the Feulgen stain. Comparison of the patterns of the Feulgen-stained karyotype with those of acetic-saline-Giemsa stain and quinacrinemustard-fluorescence demonstrates a high order of similarity among the three, with the localization of Feulgen dense bands and regions closely paralleling that of Giemsa dark and fluorescence bright bands. Since the stained substrate of the Feulgen reaction is known to be DNA, it is suggested that all three banding methods reveal the distribution of DNA or of some moiety that closely follows DNA distribution in metaphase chromosomes. The preparative procedure of the Feulgen banding method consists of a 15 to 20 minute exposure to PO₄ buffer at pH 10 and a prolonged (60–72 hrs) exposure to 12xSSC. Omission or curtailment of either step results in preparations with chromosome sets that are not karyotypable, although some stain differentiation is produced. HCl extraction prior to the preparative treatment blocks banding, but acid extraction following the preparative treatment, either that of the HCl hydrolysis of the Feulgen reaction of that of an almost fourfold extension of the standard hydrolysis time, does not obliterate bands already formed. By extrapolation from biochemical studies of chromatin, it is postulated that the localization of Feulgen dark and light stain, representing relative DNA densities, reflects the regional protein association of the DNA; the Feulgen dense regions may result from aggregation of a specific class of histones by the alkaline buffer with consequent condensation of the DNA bound to those histones; the Feulgen pale or negative regions may represent those in which non-aggregated proteins, histone and non-histone, have been solubilized in the saline incubation, rendering the DNA of those regions subject to diffusion or vulnerable to fragmentation in the Feulgen hydrolysis.     

Saggi Sul Comportamento Della Cellula Vegetale di Fronte al Reattivo di Schiff

Riassunto

Sono state fatte diverse prove per la reazione di Schiff, di Feulgen e di Bauer su tessuti vegetali. Diversi tipi di membrane, fra cui quelle lignificate, sono risultati Schiff positivi. I nuclei sono risultati Feulgen positivi, con l'eccezione dei nuclei del gametofito maturo di Crinum sp., i quali sono tutti Feulgen negativi, meno quelli antipodali. L'amido e tutte le membrane sono risultati Bauer positivi.     

Integral method for measuring the quantity of cellular DNA content by digital microphotography

Quantitative measurements of nuclear DNA content based on Feulgen reaction and the analysis of CCD images has been proposed. The measurements were performed in the monochrome CCD option (650 × 514 pixels) with a wavelength of 551 nm. The linear dependence of photomatrix element signals on the falling light was shown with a multigrade light absorption filter. The optimal microscope and camera settings and an approach for elimination of the optic blur are proposed. It was found that the contribution of background fluorescence of Feulgen-stained nuclei into the measurements was negligible. Densitometric measurements of the DNA content in blood cells of four vertebrate species (Gallus domesticus, Danio rerio, Homo sapiens, Rana arvalis) were consistent with the literature data. The precision of our approach is comparable to other known cytometry methods (). The current improvement of CCD technical parameters and the widespread use of CCD cameras in biological applications give perspectives for the development of the suggested approach for measuring the quantity of cellular DNA.     

Application of 7-amino-actinomycin D for the fluorescence microscopical analysis of DNA in cells and polytene chromosomes

Summary The cytochemical properties of a guanine-specific synthetic fluorescent analogue of actinomycin D, 7-amino-actinomycin D, have been studied in fixed and living preparations of L cells and polytene chromosomes of salivary glands ofChironomus thummi thummi andDrosophila lummei (Hackman).7-Amino-actinomycin D has been shown to bind to DNA-containing structures, thereby inducing in them a bright red fluorescence. No specific fluorescence has been found in RNA-containing structures treated with this fluorescent probe.The fluorescence pattern of some regions of polytene chromosomes with a known nucleotide composition was analysed. It has been established that 7-amino-actinomycin D induces a very weak fluorescence in GC-poor chromosome regions of theDrosophila lummei toromere structure. Data indicating a nonlinear dependence between the fluorescence intensity of a stained chromosome region and the GC content in its DNA have been obtained. The influence of DNA nucleotide composition in a chromosome region on the fluorescence of 7-amino-actinomycin D is discussed. In combination with quinacrine staining and the Feulgen fluorescence reaction, treatment with 7-amino-actinomycin D provides useful information about the distribution of GC base pairs in the chromosome region under study.     

Fluorescence fading and stabilization in cytofluorometry

Summary The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as 33258 Hoechst and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO₂ and cresylviolet-SO₂, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromoleculedye complexes generally induce fluorescence stabilization.     

Metabolic DNA in Tipula oleracea

Summary In Tipula oleracea females 2n=6+XX (Diptera) a Feulgen positive body is present in the oogonia and oocyte nuclei. This body appears in the nucleus at the oogonial divisions that precede meiosis. It gets larger by leptotene, attaining a diameter of 6 microns, and at diplotene suddenly disintegrates. By metaphase I the body is not seen.Injection of tritiated thymidine into the larvae leads to a heavy labelling of the Feulgen positive body. The body is found to synthesize DNA at a different period of time from the chromosomes, and there is an intermediate period when the synthesis of the two nuclear structures overlaps.The tritium labelled thymidine is released from the body between the third and fourth day of pupal life. At this time the yolk granules in the cytoplasm become particularly conspicuous.When the body disintegrates the labelled material becomes easily diluted. The volume of the nucleus and of the cytoplasm are sufficiently large to dilute this material in such a way that it easily becomes indistinguishable from background radiation.Spectrophotometric measurements of the body reveal that it contains four times more DNA per unit area than the chromosomes. The area of the body is 28.3 square microns and that of the chromosomes 78.5 square microns. This means that the amount of DNA in the body is of a higher order of magnitude than that found in all the chromosomes.This large amount of DNA becomes suddenly available either to the chromosomes or other cellular components. DNA can carry its own genetic information to other cellular components.This work was supported by a grant from the Swedish Natural Science Research Council.     

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

The 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.     

Tritium labelling and cytochemistry of extra DNA in Acheta

Females of Acheta domesticus were injected with H³-thymidine and H³-uridine at various stages of development in order to study DNA and RNA synthesis in the DNA body present in the oocytes. Staining with alkaline fast green, azure B and the Feulgen reaction were employed as cytochemical tests. The following main results were obtained.

1. The DNA body appears in the oogonia at interphase as a Feulgen positive spherical structure 2 microns in diameter and is seen in subsequent mitotic divisions as a slightly smaller structure of variable shape. H³-thymidine autoradiography discloses that the DNA present in this body is synthesised at a different time from the chromosomal DNA.
2. At interphase and during the early prophase of meiosis the DNA body increases in size becoming a large Feulgen positive sphere 6 microns in diameter. Small nucleoli are present within this body. The DNA of the body is complexed with histone as revealed by alkaline fast green staining. H³-thymidine labelling discloses that it is at these stages that the bulk of the DNA synthesis takes place in the body.
3. Every oocyte contains a DNA body, and no body of comparable size or shape seems to be present in the male meiotic prophase.
4. At pachytene and diplotene the DNA body acquires the appearance of a “puff”. Two zones can be distinguished inside the DNA body: (1) an inner core of DNA and an outer shell of RNA. The inner core is Feulgen positive and stains light green with azure B, the outer shell is Feulgen negative and stains purple-violet with azure B, as does the cytoplasm. From the inner DNA core many Feulgen positive fibrils radiate into the outer RNA shell. These fibrils appear unstained or slightly greenish with Azure B, forming a transparent network in a purple-violet background. This gives the body the typical appearance of a “puff”. H³-uridine incorporation reveals that the RNA synthesis occurs in the outer RNA shell of the body and in the chromosomes. RNase treatment removes the H³-uridine incorporated into these regions.
5. At the end of diplotene the DNA body starts to disintegrate. The DNA core breaks up into minor components and the outer RNA zone also begins to disintegrate. By late diplotene the whole body has vanished, releasing DNA, histone and RNA into the nucleus. Subsequently the nuclear envelope disintegrates as it regularly does at the end of prophase of meiosis.
6. The simplest interpretation of the above results is that the DNA body represents hundreds of copies of the genes of the nucleolar organizing region.

     

Increased nuclear DNA content in developing cotton fiber cells

The nuclear DNA content of developing cotton fiber cells (Gossypium hirsutum, cv. MD51ne) increases ∼24% after 2 d postanthesis (dpa). The amount of nuclear DNA at 2 dpa is 5.4 ± 0.27 pg. At 3–4 dpa it increases to 6.7 ± 0.24 pg and by 5 dpa it is 6.8 ± 0.70 pg. These values were obtained by nuclear fluorescence after staining with Hoechst 33258. Human oral squamous cell nuclei were used as a DNA standard. Nuclear DNA content increases in fibers growing on either fertilized or unfertilized ovules. The increase also is detectable in Feulgen stained nuclei using two-wavelength cytospectrophotometry. All measurements were made on isolated fiber cell nuclei using a newly developed method tailored to cotton fiber cells. The results imply that during the early stages of development fiber cell nuclei either selectively amplify certain sequences or enter S-phase replicating a portion of their genome.     

Micro‐Raman spectroscopy of algae: Composition analysis and fluorescence background behavior

Preliminary feasibility studies were performed using Stokes Raman scattering for compositional analysis of algae. Two algal species, Chlorella sorokiniana (UTEX #1230) and Neochloris oleoabundans (UTEX #1185), were chosen for this study. Both species were considered to be candidates for biofuel production. Raman signals due to storage lipids (specifically triglycerides) were clearly identified in the nitrogen‐starved C. sorokiniana and N. oleoabundans, but not in their healthy counterparts. On the other hand, signals resulting from the carotenoids were found to be present in all of the samples. Composition mapping was conducted in which Raman spectra were acquired from a dense sequence of locations over a small region of interest. The spectra obtained for the mapping images were filtered for the wavelengths of characteristic peaks that correspond to components of interest (i.e., triglyceride or carotenoid). The locations of the components of interest could be identified by the high intensity areas in the composition maps. Finally, the time evolution of fluorescence background was observed while acquiring Raman signals from the algae. The time dependence of fluorescence background is characterized by a general power law decay interrupted by sudden high intensity fluorescence events. The decreasing trend is likely a result of photo‐bleaching of cell pigments due to prolonged intense laser exposure, while the sudden high intensity fluorescence events are not understood. Biotechnol. Bioeng. 2010;105: 889–898. © 2009 Wiley Periodicals, Inc.     

Induced DNA synthesis in orchid mycorrhiza

Summary The distribution of DNA values in the cortex of mycorrhizal and non-mycorrhizal roots of Dactylorhiza purpurella and Spathoglottis plicata has been studied by Feulgen microdensitometry. Endoreduplication occurs during differentiation of cortical parenchyma. Nuclei with two or four times the normal DNA content are present in infected and adjacent uninfected cells. Autoradiography following ³H-thymidine incorporation showed that DNA synthesis is induced in fully differentiated cortical cells of S. plicata during infection by Tulasnella calospora.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.