Naegleria fowleri: Light and electron microscopy study of mitosis

DAPI and Feulgen stains were used as specific DNA markers for studying the mitosis process in Naegleria fowleri. Both DAPI and Feulgen stains reacted with DNA in the nuclei of the amoebae. Representative figures of N. fowleri mitotic nuclei with a defined arrangement according to the phase of the cell cycle were observed. A notable characteristic is that the nucleolus is present throughout the stages of mitosis. During metaphase, several deeply stained DNA condensations following an elongated pattern were observed, corresponding almost certainly to tightly grouped chromosomes. Ultrastructural observations demonstrated that the nucleus divides by cryptomitosis, a process in which the nuclear membrane does not disappear during the mitosis. Centrioles were not found, and a spindle of microtubules was observed running the length of the nucleus from pole to pole however, they did not come to a focal point.     

Tetraploid cycle in ageing solid tumours

When the mouse mammary adenocarcinoma 755 (Ca-755) reaches the plateau phase of growth, non-cycling cells with a G2-DNA content can be observed. They may belong to the diploid cell cycle but they could also be blocked in G0 or G1 of a tetraploid cycle. This hypothesis was tested in three ways: (1) non-cycling G2 nuclei were stained with a combination of Feulgen and naphthol yellow which revealed two populations, one with a low protein content and the other with a high protein content--the latter may represent nuclei ready to begin a new phase of DNA synthesis; (2) Feulgen staining and autoradiography were performed after tritiated thymidine had been administered to mice continuously: this showed that there were cells synthesizing DNA with a DNA index above 2; and (3) cells having 80 chromosomes, corresponding to the tetraploid cycle, were found almost exclusively in the plateau phase tumours. On the other hand, the use of texture and DNA parameters of the Feulgen stained nuclei showed that they were concentrated in a diploid cycle for tumours in the exponential phase of growth and were divided between a diploid and tetraploid cycle for 'plateau' cells. Neither the cause for, nor the role played by, polyploid cells is known.     

Tetraploid cycle in ageing solid tumours

Abstract. When the mouse mammary adenocarcinoma 755 (Ca-755) reaches the plateau phase of growth, non-cycling cells with a G₂-DNA content can be observed. They may belong to the diploid cell cycle but they could also be blocked in G₀ or G₁ of a tetraploid cycle. This hypothesis was tested in three ways: (1) non-cycling G₂ nuclei were stained with a combination of Feulgen and naphthol yellow which revealed two populations, one with a low protein content and the other with a high protein content– the latter may represent nuclei ready to begin a new phase of DNA synthesis; (2) Feulgen staining and autoradiography were performed after tritiated thymidine had been administered to mice continuously: this showed that there were cells synthesizing DNA with a DNA index above 2; and (3) cells having 80 chromosomes, corresponding to the tetraploid cycle, were found almost exclusively in the plateau phase tumours.
On the other hand, the use of texture and DNA parameters of the Feulgen stained nuclei showed that they were concentrated in a diploid cycle for tumours in the exponential phase of growth and were divided between a diploid and tetraploid cycle for 'plateau' cells. Neither the cause for, nor the role played by, polyploid cells is known.     

中华大蟾蜍染色体“单位线”结构的研究

适量BrdU处理中华大蟾蜍外周血淋巴细胞,常规制片后可观察到直径恒定为0.4μm的染色质纤维——单位线结构。本实验表明两栖类染色体同样具有单位线结构,并发现组成染色体两单体的姐妹单位线相伴排列,且与中期染色体有明显的形态联系。通过分析显示染色体和单位线过渡变化的分裂相,认为染色体高级结构由单位线进一步螺旋化完成,从而支持Bak模型。     

Formation and division of binucleated cells in kidney cell cultures of microtus agrestis

Summary Epithelial kidney cell cultures of Microtus agrestis contain 10 to 25% binucleated cells. Observations of living cells under the phase contrast microscope showed that binucleated cells can arise by nuclear mitosis without cytoplasmic division. When binucleated cells divide the two nuclei are highly synchronized as they enter mitosis. In mitosis the chromosomes of both nuclei combine to a common metaphase plate leading to polyploid cells. In one case a tripolar spindle was seen after formation of a metaphase by the chromosomes of the two nuclei of a binucleated cell. This tripolar mitosis resulted in one binucleated and one mononucleated cell. The DNA-content (Feulgen photometry) and the distribution of heterochromatic bodies of the nuclei were corresponding to a tetraploid, a triploid and a haploid chromosome set. This suggests the possibility of somatic segregation of complete haploid sets.Supported by the 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.     

QFH banding and DNA distribution in the macrochromosomes of the chicken Gallus domesticus

Chromosomes of Gallus domesticus were stained with rivanolum-SO2 (fluorescence Feulgen reaction) and by Hoechst 33258. Fluorescence photography was performed on a 35 mm film KN-3(KN-3) "Svema". The negatives were analyzed with the microdensitometer. The Feulgen (Fr--) and the Hoechst 3358 (H--) densitometric profiles of chromosomes showed light and dark segments along the metaphase chromosomes. The amount of DNA, as determined by the fluorescence Feulgen reaction, is not constant along the chromosome arms. Consequently, the base composition is not the only factor influencing the fluorescence of Hoechst 33258 along the chromosomes. The comparative analysis of densitometric profiles of the Hoechst 33258 and rivanolum-SO2 stained chromosomes shows that the Iq telomere band consists of GC-rich DNA (about 70% GC). The variation of DNA contents along the metaphase chromosome arms can be realized at both chromoneme and/or subchromoneme levels.     

Vegetative nuclei and differentiation of chromosomes inTrichophyton vanbreuseghemii

The stages of nuclear division were observed inTrichophyton vanbreuseghemii microcultures stained with Giemsa and by the Feulgen reaction. The course of karyokinesis is discussed. It takes the form of mitosis, modified by narrowing of the lumen of the hyphal cell and the movement of the cytoplasm. No spindle or centrioles were found. In vegetative hyphae (1.2–1.5 μm in diameter), the metaphase chromosomes were arranged lineally in a row following the direction of the long axis of the hyphal cell. The distribution of anaphase chromosomes occurs perpendicularly or obliquely to the cell walls. The chromosomes were spherical, with a diameter of about 0.3 μm. A haploid number of chromosomes (five) was found.     

Lokalisierung von rRNA-Genorten und Sekundäreinschnürungen beiVicia narbonensis undVicia sativa

Sites of 18/25S RNA genes and those of secondary constrictions have been located in metaphase chromosomes ofV. narbonensis andV. sativa by RNA/DNA in situ hybridization and Feulgen staining. InV. narbonensis the rRNA genes are located in median position on one pair of chromosomes, which is the shortest of all in the genome. InV. sativa rRNA genes are located in two pairs of chromosomes. The two heterologous sites differ markedly in the level of labeling. Strong labeling is found in a submedian position of a short pair of chromosomes. Weaker labeling is found in a median position on the longest pair of chromosomes. InV. narbonensis andV. sativa the position of the grain clusters correlate with the position of the secondary constrictions in chromosomes stained by Feulgen. The implications with respect to karyograms ofV. narbonensis andV. sativa known from the literature are discussed.

     

Advances in imaging the interphase nucleus using thin cryosections

The mammalian genome is partitioned amongst various chromosomes and encodes for approximately 30,000 protein-coding genes. Gene expression occurs after exit from mitosis, when chromosomes partially decondense within the cell nucleus to allow the enzymatic activities that work on chromatin to access each gene in a regulated fashion. Differential patterns of gene expression evolve during cell differentiation to give rise to the over 200 cell types in higher eukaryotes. The architectural organisation of the genome inside the interphase cell nucleus, and associated enzymatic activities, reveals dynamic and functional compartmentalization of the genome. In this review, I highlight the advantages of Tokuyasu cryosectioning on the investigation of nuclear structure and function. Robert Feulgen Prize 2007 Winner lecture presented at the 49th Symposium of the Society for Histochemistry in Freiburg i.Br., Germany, 26–29 September 2007.     

Some Evidence for the Specificity of the Feulgen Reaction

Carr has attacked the specificity of the Feulgen reaction on three grounds: that the chromosomes are adsorbents capable of regenerating the color of the Schiff reagent; that selectivity for the nucleus depends on destruction of cytoplasm by acid hydrolysis preceding staining; and that the reaction is not blocked by SO₂ water, as he says it should be if staining occurs by a chemical reaction. The first point was tested by staining chromosomes treated with nuclease. They were Feulgen negative, but their protein basis remained intact. The second point was tested by hydrolyzing fixed tissues, washing off solutes, drying, and comparing weight loss with controls. As differences were negligible, the fixed cytoplasm must not have been made soluble by hydrolysis. Carr's third point was not tested experimentally. It is concluded that these objections to specificity of the Feulgen reaction are not valid.     

Feulgen hydrolysis and chromosome banding in Chilocorus (Coleoptera: Coccinellidae).

Prolonged Feulgen hydrolysis of chromosomes of Chilocorus orbus Csy. and C. stigma Say produces banding patterns that are the reverse of those revealed with quinacrine; brightly fluorescing regions are unstained, but nonfluorescent regions remain relatively darkly stained. This differential reactivity at hydrolysis times that otherwise yield intense Feulgen staining confirms the need for caution in the determination of DNA values with the Feulgen reaction in material with well-defined quinacrine bands. The coincidence of DNA-specific Feulgen bands with Q-, G-, and C-bands supports the view that, in Chilocorus at least, bands reflect differences in DNA composition along the chromosome.     

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.     

Cytochemical studies on the chromatin elimination in solenobia (Lepidoptera)

Summary The chemical nature of the elimination chromatin in the first maturation division ofSolenobia was investigated with the Feulgen reaction, by staining with methyl green-pyronin. with toluidine blue before and after treatment with ribonuclease and cold perchloric acid, with the Hotchkiss periodic acid-Schiff test, and the Millon reaction.The Feulgen reaction and Hotchkiss test for polysaccharides turned out, to be negative while the tests for ribonucleic acid and protein were positive. The elimination chromatin is therefore essentially ribonucleoprotein.The elimination process is considered to be one way by which chromosomes rich in ribonucleoprotein typically present in metabolically active cells, are stripping themselves from material that may have become superfluous. Such visible shedding of material from chromosomes, though less spectacular than in the maturation division of eggs in Lepidoptera and few other insects, may be more common than is generally thought and it is suggested that the interzonal fibers found in many mitoses fall into this category.     

Differential Uptake of Tritiated Thymidine into Hetero- and Euchromatin in Melanoplus and Secale

Grasshoppers of the species Melanoplus differentialis were injected with tritium-labelled thymidine. At intervals thereafter autoradiographic stripping film was applied over Feulgen squashes and sections. In this species during early prophase of meiosis the sex chromosome forms a heterochromatic block large enough to be resolved in tritium autoradiographs. A study of the squash preparations reveals that the sex chromosome is synthesizing DNA at a different period of time from the euchromatic autosomes. Since there is a developmental sequence of spermatocyte cysts along the testicular tubes it is possible from the sections to show that the heterochromatin synthesizes DNA later than does the euchromatin. To find out whether the results obtained in Melanoplus were characteristic of heterochromatin in general, young seedlings of rye were grown in a tritiated thymidine solution and Feulgen squashes were made as for Melanoplus. In rye leaf nuclei there is a large block of heterochromatin constituted by the proximal regions of the chromosomes and a euchromatic one formed by the median and distal regions of the same chromosomes. Here also the heterochromatin synthesizes DNA at a different period of time from the euchromatin. It is concluded that in rye the asynchrony of synthesis occurs within each chromosome. Counts of silver grains over the two types of chromatin in nuclei of Melanoplus and Secale disclosed that the number of grains per unit area was two to three times higher over the heterochromatin. To check the DNA content, Feulgen photometric measurements were made of Melanoplus nuclei at the same stage. The Feulgen and grain counts agree in showing that the heterochromatin contains two to three times more DNA per unit area than the euchromatin.     

Venetian Turpentine as an Aid in Squashing and Concomitant Production of Durable Chromosome Mounts

Root tips are hydrolyzed in 1 N HCl at 60 C for 10-12 min, Feulgen stained, and macerated in a minimal amount of propiono-carmine. A drop of Venetian turpentine mounting medium (Harleco brand was used) is mixed with the carmine stain, the cover slip applied, the tissue pressed gently while observed under a dissecting microscope to spread the chromosomes, and finally firmly squashed. High quality slides of over 1 yr durability are obtained which are well suited to morphological studies, photographing under oil, or routine screening in polyploidy studies. The carmine stain in conjunction with the Feulgen aids to give contrast to chromosomes which are difficult to stain (e.g. hops) but it may be omitted for other species.     

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.     

Influence of Q- and G-banding on the Feflgen-stainability of human metaphase chromosomes

Synopsis In this paper, model experiments on chicken red blood cell nuclei are described concerning the influence of methanol-acetic acid fixation and irradiation at different wavelengths, with and without prior Atebrin staining on subsequent Feulgen-stainability. In addition, data are reported on the influence on Feulgenstainability of Giemsa-banding procedures, illumination of unstained chromosomes at 220 and 515 nm and exposure of unstained and Atebrin-stained chromosomes to illumination at 440 nm.The ASG and especially the trypsin-Giemsa technique appeared to reduce markedly Feulgen-stainability. The same holds true for Atebrin fluorescence of chromosomes. The data are discussed in relation to their implications for the assumed cause of the Q- and G-banding phenomena. Techniques are described that allow reliable Feulgen DNA measurements of individual chromosomes after application of either G- or Q-banding.     

A photographic microfluorimetric method for determining the DNA content in individual human chromosomes

A method for determination of DNA contents in individual human chromosomes has been elaborated based of the two step analysis: 1) identification of chromosomes by Q-banding; 2) photographic microfluorimetry of chromosomes after the Feulgen staining (a fluorescent variant using a Schiff-type reagent of Auramine-SO2). The DNA content in 24 human chromosomes was calculated in absolute values (fg). The data obtained are compared with the evidence published elsewhere and provided by different cytophotometric methods.     

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.