Giemsa Procedure Research Articles

Structure of the chromosomes of Scotch pine

Staining the chromosomes of Scotch pine with a simplified Giemsa procedure reveals a differentially stained heterochromatic pattern, on the basis of which four types of chromosomes can be recognized. Irregularities in the size of chromosomes as well as in the occurrence of secondary constrictions are observed. The chromatids do not separate before the centromeres divide and start to pull them apart. The use of Chlorazol black E in combination with the Giemsa procedure or with the Feulgen procedure brings out a more detailed banding pattern than by using the Giemsa procedure alone.

Genotoxic and cytotoxic effects of different types of dental cement on normal cultured human lymphocytes

In this study we have investigated the genotoxic and cytotoxic effects of eluates derived from different types of commercially available dental cements, including glass ionomer cements (GICs) ( Ketac Cem/3 M ESPE and GC Fuji I/GC Corp), resin-modified glass ionomer cements (RM-GICs) ( RelyX Luting/3 M ESPE and Vitrebond/3 M ESPE) and dual-cure resin cements (RCs) ( Variolink II/ Ivoclar-Vivadent and Panavia F 2.0/Kuraray) on normal cultured human lymphocytes. Lymphocyte primary cultures obtained from blood samples of three healthy donors were exposed to serial dilutions of eluates derived from specimens of each material tested. Metaphases were induced with phytohaemagglutinin, collected after 72 h treatment by use of colchicine and stained according to the fluorescence plus giemsa (FPG) procedure. Preparations were scored for sister chromatid exchange (SCE) and chromosomal aberrations (CAs), while the proliferation rate index (PRI) was also calculated. Our results show that eluates derived from the RM-GICs and RCs caused severe genotoxic effects by significantly increasing the frequencies of SCEs and CAs in cultures of peripheral blood lymphocytes and by decreasing the relevant PRI values in a dose-dependent manner, whereas the two GICs caused only minor cytogenetic effects. Eluates of the two RM-GICs (Vitrebond and RelyX) were also very cytotoxic, as the first serial dilutions of both materials caused a complete mitotic arrest in lymphocyte cultures. Overall, the degree of genotoxicity and cytotoxicity caused by dental cements decreased as follows: Viterbond > Rely X > Panavia F 2.0 > Variolink II > Ketac Cem = GC Fuji I. These results indicate that different types of dental cement differ extensively in their genotoxic and cytotoxic potential and their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. Although these results cannot be directly extrapolated to the clinical situation, the potential occurrence of adverse effects caused by the RM-GICs and RCs tested in this study should be considered when making a clinical decision about dental cements.

Sister-chromatid exchange, chromosomal aberrations and delays in cell-cycle kinetics in human lymphocytes induced by dental composite resin eluates

We have investigated eluates derived from commercially available composite resin-based materials used for direct (Tetric Ceram/Ivoclar-Vivadent, Simile/Pentron, Filtek Z-250/3M ESPE) and indirect (Adoro/Ivoclar-Vivadent and Conquest Sculpture/Pentron) dental restorations, with respect to their genotoxic effects on human peripheral lymphocytes. Primary lymphocyte cultures obtained from blood samples of three healthy donors were exposed to eluates of freshly cured specimens of all the materials tested. Metaphases were induced with phytohaemagglutinin, collected after a 72-h treatment using colchicine and stained with the Fluorescence Plus Giemsa (FPG) procedure. Preparations were scored for sister-chromatid exchange (SCE) and chromosomal aberrations (CAs). The proliferation rate index (PRI) and the mitotic index (MI) were also calculated. Our results show that eluates derived from the three direct composites (Filtek Z-250, Simile and Tetric Ceram) increased the frequencies of SCE and CAs and markedly reduced PRI and MI. Tetric Ceram's eluate, being the most genotoxic of all eluates tested, increased the frequencies of SCE up to 24.40 per cell (control, 9.87 per cell) and of CAs up to 424 per 100 metaphases scored (control, 5). Moreover, it caused a pronounced decrease of the PRI down to 1.31 (control, 2.44) and of the MI down to 9.8‰ (control, 19.2‰). In contrast, eluates derived from the laboratory-processed composites (Adoro and Conquest Sculpture) induced much less cytogenetic damage. Overall, the degree of genotoxicity and cytotoxicity decreased as follows: Tetric Ceram > Filtek Z-250 > Simile > Adoro = Conquest Sculpture. These results indicate that composite resins used for direct and indirect dental restorations differ extensively in their cytotoxic and genotoxic potential and in their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. This underlines the impact of improved polymerization with respect to their biological behavior.

Blood Treatment and Yield of Apoptotic Leukocytes following Exercise

Detection of apoptosis is difficult in vivo because of the rapid clearance of apoptotic cells via phagocytosis. Since the apoptotic process can occur within minutes rather than hours, it is possible that the percentage yield observed may be affected by the technique used to assess apoptosis, especially if using various time-consuming cell isolation procedures. PURPOSE: to examine the effect of blood treatment on the yield of post-exercise apoptotic leukocytes (apoptotic index: AI). METHODS: Twelve moderately trained subjects (mean±SE age=22.7±0.7yr, VO2max= 53.1±1.3ml·kg-1·min-1) performed two 40 min treadmill exercises at ∼70% VO2max: running on a level grade (L) and at −10% downhill (DH). Blood was sampled from an antecubital vein before (PRE) and immediately (POST), 2h, 24h, and 48h following each run. Samples were partitioned into two treatments: 1) whole blood smear (WB) made immediately after the sample was obtained, stained using the May Grünwald Giemsa procedure, with a minimum of 200 leukocytes evaluated under a light microscope for morphological identifcation of apoptosis, 2) leukocyte isolation (ISO) using an ammonium chloride technique and AI measured using the TUNEL assay with flow cytometry. Data were analyzed using a 2-way repeated measures ANOVA with post hoc Tukey tests. RESULTS: A significantly higher (3.2–5.6 fold) AI was found when using WB vs ISO (p< 0.05 at PRE, P<0.01 for the remaining time points). Within trial, both treatments produced the same statistical outcome following DH, i.e., AI was significantly elevated above PRE at all time points (p< 0.05 at 48h ISO, P<0.01 for the remaining time points). Following L, AI was significantly elevated above PRE at POST, 2h, and 24h using WB, but significantly elevated above PRE only at POST and 2h using ISO (p<0.01). Between trials, AI was significantly higher in DH vs L at 2h (P<0.05), 24h and 48h (P<0.01) using WB, but only at 24h (P<0.01) and 48h (P<0.05) using ISO. CONCLUSIONS: Using whole blood yielded a significantly higher leukocyte AI compared to a technique using isolated cells, perhaps due to an enhanced apoptotic response to ammonium chloride lysing and/or the amount of time needed to carry out the isolation process. Differential blood treatment also affected statistical outcomes.

Blood Treatment Influences the Yield of Apoptotic Lymphocytes after Maximal Exercise

No systematic investigation has been reported assessing the effect of cell isolation processes on postexercise apoptosis. Therefore, the effect of cell isolation procedures on apoptosis was evaluated in this study. Untrained healthy individuals participated (N=13). Blood samples obtained at rest and immediately after an incremental exercise test to exhaustion were partitioned into three treatments: 1) whole blood smears made immediately after the sample was obtained (WB), 2) cells subjected to density-gradient isolation before smears were made (ISO), and 3) samples allowed to sit at room temperature (i.e., time-treated) before centrifugation and smearing (TT). Blood smears were stained using the May-Grünwald Giemsa procedure and lymphocytes were evaluated under a light microscope for characteristic features of apoptosis. Data were analyzed using a 2x3 ANOVA. A significant interaction effect existed (P<0.0001) such that at rest, no difference was detected in the amount of lymphocyte apoptosis among WB, ISO, or TT samples. However, after exhaustive exercise, the amount of apoptotic lymphocytes was significantly greater in WB compared with ISO and TT samples (P<0.0001). Lymphocyte isolation results in a significant decrease in the percent of apoptotic lymphocytes after exhaustive exercise. This reduction is likely due to the time needed to isolate cells, rather than the isolation process itself. Because apoptosis is a time-sensitive process that occurs within minutes rather than hours, the length of time from initial sampling to the preparation of cells for assessment of apoptosis is critical and should be considered in future exercise studies.

The chromosomes of Rodents of the Republic of Benin (West Africa): 2. Sciuridae and Thryonomyidae

The karyotype has been studied in 2 species of Rodents, the Redless squirrelFunisciurus anerythrus (Sciurognathi, Sciuridae) and the Cane ratThryonomys swinderianus (Hystricognathi, Thryonomyidae), from crop lands and woodlands of the Republic of Benin (West Africa). Chromosomes were obtained by bone marrow method and stained both by standard Giemsa and NOR banding procedures. T.swinderianus shows a2n = 44 (NF = 86) complement. Chromosomes are all biarmed except for one pair acrocentrics. Nuclear Organising Regions (NORs) are located on one submetacentric pair coinciding with a secondary constriction.F. anerythrus shows a2n = 38 (NF = 62) complement. The karyotype is composed of 7 acrocentric chromosomal pairs and 12 biarmed chromosomal pairs. The karyotype morphology of the species was discussed within the context of Old World Hystricognathi and African Sciuridae comparative cytogenetics.

Relationship of chromocenter number, size, and density to plant biomass

SUMMARYA variety of plant species and cultivars were stained for chromocenters using the barium hydroxide, sodium saline citrate (SSC), Giemsa procedure (DNA denaturation, incubation-renaturation, staining), in order to test the hypothesis that larger (biomass) species have larger, more numerous, and more intensely-staining chromocenters. In all species and cultivars examined, results confirmed the hypothesis. Since chromocenters are believed to be composed primarily of heterochromatin/repetitive DNA (which has been postulated to shorten the cell cycle), it is likely that chromocenters modulate species size/biomass. Among closely related species, larger/more biomassive cultivars contained larger, more numerous, and more intensely-staining chromocenters than smaller/less biomassive cultivars.

Identification of Acanthamoeba in bronchoalveolar lavage specimens.

Acanthamoeba species infect humans occasionally and act as opportunistic pathogens in immunocompromised individuals. This study demonstrates the application of cytocentrifugation as an aid to identification of Acanthamoebae. In addition, certain staining procedures clearly optimized visualization of characteristic amoebic features. This was demonstrated by adding amoebae from laboratory cultures to bronchoalveolar lavage specimens. In preparations stained by the Papanicolaou, trichrome, or hematoxylin-eosin (H&E) procedures, the discrete deeper staining nucleolus was the most distinctive feature. The vacuolated cytoplasm also aided in the identification of amoebae. These features were less apparent and often distorted following staining of Acanthamoeba species with the Hema III and Giemsa procedures.

Understanding microwave-stimulated Romanowsky-Giemsa staining of plastic embedded bone marrow

Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky--Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky--Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.

Alder Anomaly Accompanied by a Mutation of the Myeloperoxidase Structural Gene

In this paper, a third form of Alder anomaly is described. In addition to the morphological changes which are characteristic of this anomaly, the peroxidase of the neutrophil polymorphonuclear leukocytes (NPNL) is abnormal in its properties. It is resistant to the fixation step in the May-Grünwald Giemsa procedure and is similar, therefore, to the eosinophil peroxidase. In the phenotype observed, 2 genetic changes are probably involved: one is a change in the regulation of NPNL granulation, the other is a mutation in the myeloperoxidase structural gene. In the light of these new findings, 3 forms of the anomaly are now known: the original Alder anomaly, in which the abnormal granulation, particularly in the granulocytes, is not linked with any hereditary disorder; the Alder-Reilly form, in which in addition to the abnormal granulation the leukocytes also contain inclusion bodies, the phenomenon sometimes being associated with mucopolysaccharidosis (gargoylism), and the third form of anomaly described here, where-apart from the abnormal granulation-the myeloperoxidase behaves differently, resembling that of eosinophils in its resistance to methanol fixation.

The involvement of nucleosomes in Giemsa staining of chromosomes. A new hypothesis on the banding mechanism.

A new hypothesis is proposed on the involvement of nucleosomes in Giemsa banding of chromosomes. Giemsa staining as well as the concomitant swelling can be explained as an insertion of the triple charged hydrophobic dye complex between the negatively-charged super-coiled helical DNA and the denatured histone cores of the nucleosomes still present in the fixed chromosomes. New cytochemical data and recent results from biochemical literature on nucleosomes are presented in support of this hypothesis. Chromosomes are stained by the Giemsa procedure in a purple (magenta) colour. Giemsa staining of DNA and histone (isolated or in a simple mixture) in model experiments results in different colours, indicating that a higher order configuration of these chromosomal components lies at the basis of the Giemsa method. Cytophotometry of Giemsa dye absorbance of chromosomes shows that the banding in the case of saline pretreatment is due to a relative absence of the complex in the faintly coloured bands (interbands). Pretreatment with trypsin results in an increase in Giemsa dye uptake in the stained bands. Cytophotometric measurements of free phosphate groups before and after pretreatment with saline, reveal a blocking of about half of the free phosphate groups indicating that a substantial number of free amino groups is still present in the fixed chromosomes. Glutaraldehyde treatment inhibited Giemsa-banding irreversibly while the formaldehyde-induced disappearance of the bands could be restored by a washing procedure. These results correlate with those of biochemical nucleosome studies using the same aldehydes. Based on these findings and on the known properties of nucleosomes, a mechanism is proposed that explains the collapse of the chromosome structure when fixed chromosomes are transferred to aqueous buffer solutions. During homogeneous Giemsa staining reswelling of the unpretreated chromosome is explained by insertion of the hydrophobic Giemsa complex between the hydrophobic nucleosome cores and the superhelix DNA. Selective Giemsa staining of the AT-enriched bands after saline pretreatment is thought to be due to the, biochemically well-documented, higher affinity of arginine-rich proteins present in the core histones for GC-enriched DNA, which prevents the insertion of the Giemsa complex in the interbands. Production of Giemsa bands by trypsin pretreatment can be related to the action of this enzyme on the H1 histones and subsequent charge rearrangements.(ABSTRACT TRUNCATED AT 400 WORDS)

IDENTIFICATION OF PARTIAL TRISOMY 13 SYNDROME BY DNA REPLICATION STUDIES

The Giemsa stainability of chromosomal regions containing late replicating DNA can be suppressed or enhanced by incorporation of BUdR into the DNA at the end or beginning of the S period. A terminal pulse of BUdR reveals a uniquely informative pattern of chromosomal bands coupled with specific decondensation of late replicating regions. These techniques provide new information for the clinician and cytogeneticist in precise identification of heretofore undecipherable “de novo” structural chromosomal rearrangements with partial aneuploidy. Our patient was a one month old male with multiple congenital anomalies including postaxial polydactyly, heart defect and renal anomaly. Conventional chromosomal analysis revealed a 46, XY, Dq+ karyotype, while Trypsin G-banding suggested an inverted duplication of the distal long arm of chromosome 13 (i.e. 46, XY, inv dup (13q) (ql4→q34). Chromosomes of both parents are normal. Bands q21 and q31 of chromosome 13 are late replicating and would be included in the presumed duplication. Employing a modified “33258 Hoechst” Giemsa procedure, and chromosomes prepared for enhancement and suppression of stainability of late replicating regions, we observed the predicted four bands of late replication on the abnormal D group chromosome, thus allowing confirmation of the diagnosis of partial Trisomy 13 Syndrome.

A modified technique for combination of cytochemistry and radioautography of haemic cells.

Cultured mouse bone marrow and human blood cells were labelled with 3H-thymidine, smeared, and stained, using peroxydase reagents or the May-Grünwald Giemsa procedure. The slides were dipped in a approximately 12% solution of polyvinylchloride, bleached by a photographic fixer and processed for liquid emulsion radioautography. This method yielded technically very satisfactory radioautographs of pre-stained smears.

Selective staining of Y chromosomal loops in Drosophila hydei, D. neohydei, and D. eohydei.

Modified Giemsa procedures have been developed which elicit differential and highly selective staining of individual Y chromosomal lampbruch loops in spermatocyte nuclei of Drosophila hydei, D. NEOHYDEI, AND D. eohydei. In all three species the Y loop pair known as the "clubs" stains a brilliant dark red with Giemsa at pH 10. With the same treatment other loop pairs either remain unstained, e.g. the "threads", or show a differentiation between light blue and pink staining material, e.g. "pseudonucleolus" and "cones" in D. hydei and D. eohydei. With eosin at pH 2.8 the "threads" in D. hydei can be stained intensely, as well as one matrical component of th "pseudonucleolus". Pretreatment with RNase or TCA removes all stainability from the Y loops with Giemsa at Ph 10. TCA treatment enhances eosin staining at pH 2.8. These and other variations of Giemsa may be utilized to establish homologies between Y loops in different species. The molecular basis of the staining reactions remains to be elucidated.

Heterochromatin diversity in Cymbidium, and its relationship to differential DNA replication

1. 1. DNA was extracted from aseptical cultures of protocorms of the orchid Cymbidium and analysed by thermal denaturation. The denaturation profiles revealed an AT-rich fraction of about 18% of total DNA. 2. 2. Mitotic chromosomes and diploid and endopolyploid nuclei of in vitro cultured protocorms and root tips were differentially stained with quinacrine (Q), 33258 Hoechst (H) and a novel compound 4′-6-diamidino-2-phenylindole (DAPI) [10], as well as by the Giemsa C-banding technique. The centromeric regions display very bright fluorescence with all three fluorochromes and stain intensely following the Giemsa procedure. It is proposed that the AT-rich fraction of the Cymbidium DNA is located within the centromeric heterochromatin. 3. 3. In interphase nuclei differential Q, H, and DAPI fluorescence both within and between the chromocenters occurs. In nuclei with enlarged chromocenters, i.e. with amplified DNA in heterochromatin [2], the increased size of chromocenters is mainly caused by enlargement of the less brightly fluorescing fractions of the heterochromatin. The proportion of the very brightly fluorescing heterochromatin is similar in all nuclei (about 7%). 4. 4. A comparison of nucleolar size and differential Giemsa staining of the nucleolus organizers showed that there is no disproportional increase of nucleoli and nucleolus organizing heterochromatin during endopolyploidization. That means, there is no indication for amplification of ribosomal DNA. 5. 5. Electron micrographs particularly of heterochromatin-rich nuclei revealed areas of different chromatin density within and between the chromocenters. However, the differences in fiber packaging density are much smaller than the observed differences in fluorescence brightness. 6. 6. The data obtained are interpreted as evidence for differential replication of AT-rich and non-AT-rich heterochromatin. It is suggested that DNA amplification [2, 4] is restricted to a non-AT-rich component which apparently is located neither in the brightly fluorescent centromeric nor in the nucleolus-associated heterochromatin.

Sister chromatid differentiation and exchanges in adult mudminnows (Umbra limi) after in vivo exposure to 5-bromodeoxyuridine.

An in vivo system for the detection of sister chromatid exchange (SCE) in the central mudminnow, Umbra limi, is presented. Sister chromatid differential (SCD) and SCE were demonstrated by fluorescent and Giemsa procedures 5 to 6 days after the fish were injected with 500 mug/g of BrdU. The exchange rate was found to be 2.64 SCEs metaphase in the intestines and 2.42 SCEs/metaphase in the gills. SCE analysis in U. limi should be a useful tool for measuring the mutagenicity of water-borne chemicals.

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.

New selective Giemsa technique for human chromosomes, Cd staining.

AFTER the introduction of the quinacrine fluorescence method1, several Giemsa staining techniques have been developed for karyotype analysis of human chromosomes. Pardue and Gall2, originally noticed a denser staining of centromeric regions of chromosomes after in situ hybridisation of mouse chromosome preparations with mouse satellite DNA followed by Giemsa staining. This initial approach was modified by Arrighi and Hsu3 who omitted DNA hybridisation. Later, Sumner et al.4 left out treatment with RNase and HC1 as well. Finally, McKenzie and Lubs5 treated chromosomes with HCl and 2×SSC only. These modified Giemsa procedures all produce densely stained regions of one or both chromosome arms close to the centromere. These C-band procedures also stain the secondary constriction of chromosome 1, 9 and 16, as well as the distal part of the Y chromosome. Satellites that stain brightly by Q-band techniques are also revealed by these methods.

Giemsa banding of meiotic chromosomes

Applying a Giemsa staining technique to the meiotic chromosomes of Anemone blanda demonstrates that Giemsa bands similar to those seen in the mitotic chromosomes are discernible at all the principal stages of meiosis. The bands are not a product of the Giemsa procedure since they can be seen in unstained preparations using phase-contrast optics as chromocentres in interphase nuclei and as condensed regions in prophase chromosomes. That the bands seem to be permanent features of the nucleus, whether it is dividing or otherwise is an important consideration for understanding their nature and function. Bands and chiasmata do not coincide indicating on the one hand that chiasmata are not responsible for differences in banding patterns and on the other hand that the conservation of bands is an indication that they are either inert regions or specialised regions with considerable adaptive significance. These alternatives can only be resolved by genetical studies of the banding phenomena.

Demonstration of pathogenic fungi in formalin-fixed tissues by immunofluorescence.

This study was designed to improve current methods for demonstrating fungi in sections of formalin-fixed, paraffin-embedded tissue by the fluorescent antibody technic. The findings in this study indicate that digestion of sections in 1.0% trypsin solution for 1 hr. at 37 C. before conjugates are applied is a single, inexpensive procedure that enhances considerably the staining of Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, and Sporotrichum schenckii. The results of this study also demonstrate that these fungi can be stained by the fluorescent antibody technic in tissue sections that have been stained previously by the hematoxylin and eosin, the Brown and Brenn, and the Giemsa procedures. They cannot be stained, however, in sections that have been stained previously by the Gomori methenamine-silver nitrate, the periodic acid-Schiff, or the Gridley procedures. Apparently, the oxidation of polysaccharides in the walls of these fungi by the periodic acid or the chromic acid alters the antigenicity of these organisms so that they no longer react with the labeled antibodies. The addition of dimethyl sulfoxide to conjugates in a ratio of 1:9 or 1:14 was found to improve the fluorescent antibody staining results obtained with these fungi in formalinized tissue sections. This improvement is the result of enhancement of contrast between stained organisms and the background.