Conventional Giemsa Staining Research Articles

HELICOBACTER PYLORI INFECTION AND TREATMENT OUTCOME IN AN URBAN AUSTRALIAN POPULATION

To examine the rate of Helicobacter pylori infection in a group of symptomatic patients using rapid urease test and antral histology and report on their treatment outcomes. From a single-surgeon series in a tertiary teaching hospital, 200 consecutive symptomatic patients undergoing their first diagnostic upper gastrointestinal endoscopy during 2003-2004 were included in this retrospective audit. Campylobacter-like organism (CLO) test results were compared with the results of conventional histological examination plus Giemsa staining. Eradication rates and demographic data were collected. One hundred and seventy-nine of 200 (89.5%) patients had antral biopsies for histology and for CLOtest. The CLOtest was positive in 31% (56/179) of patients. Thirty-two of 56 (57%) patients had an early-positive CLOtest (<24 h). The remaining 43% (24/56) of patients had a late-positive CLOtest (>24 h). Histological examination of the antral biopsy showed evidence of H. pylori in 28/56 (50%) patients with a positive CLOtest. H. pylori eradication therapy was prescribed in 51/56 (91%) patients with a positive CLOtest. Forty-five of 51 (88%) of these patients had a subsequent urea breath test. Urea breath test was positive in 17/45 (29%) patients after H. pylori eradication therapy was prescribed. Rate of H. pylori in this series of symptomatic patients was 31%. As nearly half of the CLOtests became positive after 24 h, we suggest that the CLOtest should be re-examined 24 h after gastroscopy. Standard antral histology and CLOtest were in agreement in only 50% of cases, suggesting that the CLOtest is a more sensitive test. The failure rate of 29% for initial H. pylori eradication therapy reinforces the need for follow up after treatment.

Karyotypes of Rana tagoi Okada with diploid number 28 in the Chausu Mountains of the Minamishinshu district of Nagano Prefecture, Japan (Anura: Ranidae)

Karyotypes of Tago’s brown frog Rana tagoi from the Chausu mountains in Minamishinshu of Nagano Prefecture were examined by conventional Giemsa staining, C-banding and late replication (LR)-banding. Chromosome number was 2n = 28 in all cases. The 28 chromosomes consisted of four pairs (1–4) of large biarmed chromosomes, two pairs (5–6) of telocentric chromosomes and eight pairs (7–14) of small biarmed chromosomes. Chromosome pair 11 had a secondary constriction on the long arm. In females, the C-band on the long arm of chromosome pair 6 was detected in both homologs, but was absent from the arms of the homologs of chromosome pairs 5 and 9. In males, C-bands were found in the long arms of both homologs of chromosome pairs 5 and 6, were present only in one homolog of chromosome pair 5 for certain male specimens and found in only one homolog of chromosome pair 9. Specimens of R. tagoi (2n = 28) should thus have two pairs of telocentric chromosomes to provide the same number of chromosome arms, these originating quite likely from chromosome pair 1 in the 26-chromosome specimens by centric fission. Heteromorphic sex chromosomes of the XX-XY type in R. tagoi (2n = 28) in the Chausu mountains were identified. Karyotypes of tail-tip cells from a hybrid tadpole between female R. tagoi (2n = 26) from the Hinohara village in Tokyo and male R. tagoi (2n = 28) from the Chausu mountain population were examined by squash preparation. Chromosome number was 2n = 27 in all tadpoles. The 27 chromosomes consisted of one chromosome set of R. tagoi (2n = 28) and one of R. tagoi (2n = 26).

Diversity of Dilepididae (Cestoda: Cyclophyllidea) revealed by cytogenetic analysis

Karyotypes of three dilepidid species: Molluscotaenia crassiscolex, Anomotaenia bacilligera and Dilepis undula, which have not been recorded previously, were studied using conventional Giemsa staining and comparative karyometric analysis. Twelve small biarmed chromosomes were observed in mitotic cells of M. crassiscolex, 16 biarmed chromosomes of gradually decreasing size were found in cells of A. bacilligera, while 18 elements were characteristic for D. undula. These data, together with information available in literature, prove the heterogeneity and possible polyphyletic nature of the family Dilepididae.

Evaluation of spectral karyotyping (SKY) in biodosimetry for the triage situation following gamma irradiation

Purpose: Biological dosimetry in an acute triage situation of radiation exposure is traditionally performed by scoring unstable dicentric chromosomal aberrations after conventional Giemsa staining, and more recently also by detection of chromosomal translocations after chromosome painting by fluorescence in situ hybridization (FISH). By spectral karyotyping (SKY) each chromosome can be painted in an individual colour, permitting the scanning for structural aberrations throughout the genome in each individual metaphase. Here we have evaluated the performance of SKY analysis in a simulated triage situation after gamma irradiation.Materials and methods: Peripheral leukocytes were irradiated by 60Co (0 – 5 Gy) and analysed by SKY, Giemsa staining and FISH painting of chromosomes 1, 2, and 3.Results: At 1 Gy and higher doses, dicentric aberrations (Dic+) as well as classical one- and two-way translocations were found in increasing and dose-dependent frequencies by SKY. The frequency of dicentrics detected by Giemsa was found to be significantly higher than the total aberrations detected by SKY (p<0.001), but did not differ significantly from that of FISH painting. The difference was mainly attributable to the low sensitivity of SKY to detect Dic+ following frequent lack of acentric fragments with matching chromosomal composition.Conclusions: The findings anticipate that radiation induced chromosomal aberrations may be more complex than expected from conventional and single chromosome painting analyses. While conventional Giemsa staining was found to be the method of choice for the triage situation, it is expected that extended SKY analysis will add to the knowledge of underlying mechanisms for irradiation associated chromosomal aberrations.

Intra- and inter-individual chromosome variation in Hoplerythrinus unitaeniatus (Pisces, Erythrinidae): a population from the Brazilian São Francisco river basin

Chromosomal studies were carried out on a population of the fish Hoplerythrinus unitaeniatus (jeju) from Prata river in the Sao Francisco river basin, Minas Gerais, Brazil using conventional Giemsa staining, C-banding, silver nitrate nucleolar organizer region (Ag-NOR) staining and fluorescent in situ hybridization (FISH) with 18S and 5S rDNA probes. We found a high degree of inter- and intra-individual variability with the identification of 2n = 50, 2n = 51 and 2n = 52 karyotypes at nearly the same frequency within the population. Intra-individual variation in chromosomal morphology and, consequently, karyotype formulae was also observed, chiefly in the specimens with 2n = 50 and 2n = 52 chromosomes. Ag-NORs and 18S rDNA sites also showed numerical and chromosomal variation similar to that found for the 5S rDNA sites. Some putative hypotheses are considered in order to explain these results.

Karyotype Differentiation in Chromaphyosemion Killifishes (Cyprinodontiformes, Nothobranchiidae). I: Chromosome Banding Patterns of C. Alpha, C. Kouamense and C. Lugens

The karyotypes of three recently described species of Chromaphyosemion, namely C. lugens, C. alpha and C. kouamense, were analysed using conventional Giemsa staining, C-banding and sequential banding (fluorescence banding with 4', 6-diamidino-2-phenylindole (DAPI) and Chromomycin A(3) (CMA(3)), C-banding, AgNO(3)-staining). Diploid chromosome numbers ranged from 2n = 36 in C. lugens to 2n = 38 in C. alpha and C. kouamense. The karyotype of C. lugens consisted of one pair of metacentric (m) and 17 pairs of telocentric (t) chromosomes, that of C. alpha was composed of one pair of submetacentric (sm), six pairs of subtelocentric (st) and 12 pairs of t chromosomes, and that of C. kouamense comprised five pairs of st and 14 pairs of t chromosomes. In addition to the differences in karyotype structures and/or chromosome numbers, the karyotypes of the examined species differed with respect to NOR phenotype and distribution and base composition of heterochromatin. No heteromorphic sex chromosomes were detected in any of the species. Our findings provide cytotaxonomic evidence for the species distinctness of C. alpha, C. kouamense and C. lugens whose descriptions were based primarily on external morphology.

Karyotypes of two American field crickets: Gryllus rubens and Gryllus sp. (Orthoptera: Gryllidae)

AbstractThe karyotypes of Gryllus rubens (Scudder) and Gryllus sp. collected in the US were studied using conventional Giemsa staining and two differential staining methods. Both species had a chromosome complement of 2n = 28 + XX/XO, and the X chromosome was large and metacentric. In addition, nucleolus organizer regions (NOR) were detected in the short arm of one pair in the two species, and the NOR showed variation in size. The two species had species‐specific chromosome configuration and C‐banding patterns. In Gryllus sp., the chromosome configuration showed polymorphism in size and type among individuals, and the distal C‐bands of Gryllus sp. were larger than those of G. rubens.

EVALUATION OF THE POLYMERASE CHAIN REACTION ANALYSIS FOR DIAGNOSIS OF FALCIPARUM MALARIA IN DELHI, INDIA

Plasmodium falciparum infections are frequently fatal if untreated and hence need to be diagnosed and treated early. Malaria diagnosis, with conventional Giemsa staining as a gold standard, has had several limitations. New rapid and accurate methods are needed for diagnosis. In this study, polymerase chain reaction (PCR) analysis specific for diagnosis of P. falciparum was evaluated. For the study, blood samples were collected from 310 patients suspected of having malaria. PCR analysis for P. falciparum from venous blood and at the same time Giemsa staining of thick and thin blood smears was done. A total of 160 (51.6 %) samples were positive for malarial parasite of which 63 (39.4 %) were positive for P. falciparum by Giemsa staining while 61 (38.1 %) were positive for P. falciparum by PCR analysis. Giemsa staining was time consuming, laborious and may give poor results in cases with low parasitaemia. The PCR analysis for P. falciparum was able to detect 3 cases of low parasitaemia missed initially on Giemsa staining, was 96.8 % sensitive, 100% specific but was very costly, needed a lot of practice and standardization and was time consuming. PCR analysis can be used to supplement the conventional Giemsa staining for reliable diagnosis of falciparum malaria especially in cases with low parasitaemia.

Evaluation of the polymerase chain reaction analysis for diagnosis of falciparum malaria in Delhi, India

Plasmodium falciparum infections are frequently fatal if untreated and hence need to be diagnosed and treated early. Malaria diagnosis, with conventional Giemsa staining as a gold standard, has had several limitations. New rapid and accurate methods are needed for diagnosis. In this study, polymerase chain reaction (PCR) analysis specific for diagnosis of P. falciparum was evaluated. For the study, blood samples were collected from 310 patients suspected of having malaria. PCR analysis for P. falciparum from venous blood and at the same time Giemsa staining of thick and thin blood smears was done. A total of 160 (51.6 %) samples were positive for malarial parasite of which 63 (39.4 %) were positive for P. falciparum by Giemsa staining while 61 (38.1 %) were positive for P. falciparum by PCR analysis. Giemsa staining was time consuming, laborious and may give poor results in cases with low parasitaemia. The PCR analysis for P. falciparum was able to detect 3 cases of low parasitaemia missed initially on Giemsa staining, was 96.8 % sensitive, 100% specific but was very costly, needed a lot of practice and standardization and was time consuming. PCR analysis can be used to supplement the conventional Giemsa staining for reliable diagnosis of falciparum malaria especially in cases with low parasitaemia.

New contributions to the cytotaxonomy of tiger beetles (Coleop-tera, Cicindelidae) from the Afrotropical Region: cytogenetic characterization of Prothyma concinna, Elliptica lugubris and Ropaloteres cinctus

Metaphases from germinal tissue of three Afrotropical species of tiger beetles occurring in Guinea-Bissau were analysed using conventional Giemsa-staining. The species showed distinct karyotypes an...

Cytogenetic and sequence comparison of adultPhyllodistomum(Digenea: Gorgoderidae) from the three-spined stickleback with larvae from two bivalves

Due to the low informative value of available morphological characters, cytogenetic and molecular methods, based on rDNA sequencing, were used to characterize adult and larval stages of Phyllodistomum spp. Species studied have 18 chromosomes with comparable absolute and relative lengths. Conventional Giemsa staining and karyometric analysis revealed clear differences in chromosome morphology of larval Phyllodistomum spp. infecting two bivalve host species, Sphaerium corneum and Pisidium amnicum. However, karyotypes of adult P. folium from three-spined sticklebacks and larval stages from S. corneum appear almost identical both with respect to the relative lengths and centromeric indices of the corresponding chromosome pairs. The entire internal transcribed spacer (ITS) region (ITS-1, 5.8S and ITS-2) and the D1-D3 region of 28S gene were sequenced and compared. Again, sufficient differences were observed between larval Phyllodistomum spp., while adult P. folium and larvae from S. corneum showed a high level of similarity. So, both cytogenetic and molecular data support the suggestion that they represent developmental stages of the same species. The results were compared with published data obtained by cytogenetic and molecular studies on the other Phyllodistomum species. Differences revealed in karyotype and rDNA sequences leads to the conclusion that the cercariaeum of P. folium sensu Sinitsin, 1905 could not be regarded as the larva of adult P. folium from three-spined stickleback.

Karyology of the toadfish Porichthys plectrodon (Jordan and Gilbert, 1882) (Batrachoididae) from Margarita Island, Venezuela

This paper reports the results of cytogenetic analyses carried out on Porichthys plectrodon using conventional Giemsa staining, C-banding and silver staining techniques. A diploid chromosome count of 2n=44 was observed, consisting of 8 metacentric, 10 submetacentric, 6 subtelocentric and 20 acrocentric chromosomes. Differences in length made it possible to identify homologous chromosomes within the metacentric group. Constitutive heterochromatin was distributed as large pericentromeric blocks in pairs 1 and 2, while the rest of the chromosomes were marked in centromeric regions, some more conspicuously than others. One pair of small-sized acrocentric NOR-bearing chromosomes (21) was identified by the nucleolar regions located terminally on their short arms.

Chromosome analysis and rDNA FISH in the stag beetle Dorcus parallelipipedus L. (Coleoptera: Scarabaeoidea: Lucanidae).

In the present work the chromosome complement (2n = 18; 8AA + XY) of the stag beetle Dorcus parallelipipedus L. (Scarabaeoidea: Lucanidae) is analyzed using conventional Giemsa staining, banding techniques and ribosomal fluorescent in situ hybridization (rDNA FISH). rDNA FISH remains the unique tool for providing a clear-cut identification of Nucleolar Organizer Regions (NORs) when conventional banding methods such as silver- and CMA3-staining proved to be inadequate. The dull, homogeneous CMA3 fluorescence of all chromosomes indicates the absence of markedly GC rich compartmentalized regions in D. parallelipipedus genome. Silver impregnation inadequacy in detecting NOR regions is to be sought in the unusual extensive silver stainability of heterochromatic material which, on the contrary of what stated for vertebrates, seems to be a common feature in Scarabaeoidea species.

Chromosome aberration and lipid peroxidation in chromium-exposed workers

Chromosome aberration frequency and lipid peroxidation levels were analysed to investigate their efficacy as biological markers for monitoring the genotoxicity and oxidative damage in Korean chromium (Cr)-exposed workers. Fifty-one Cr-exposed workers and 31 age-matched controls in ten chrome-plating plants were sampled. The Cr level was measured in the workers’ blood and urine, and in the ambient air at the workplaces. The conventional Giemsa staining method and fluorescence in situ hybridization (FISH) technique were used for chromosome aberration analysis. Spectrum green whole chromosome paint specific for chromosome 4 was used in the FISH procedure. As for lipid peroxidation, malondialdehyde (MDA) was measured in the blood plasma as thiobarbituric acid-reactive substances (TBARS). The blood Cr concentration was statistically correlated with both the frequency of chromatid exchange and the total frequency of chromosome/chromatid breaks and exchanges, as detected by the Giemsa staining. Meanwhile, the frequency of translocation, as detected by the FISH technique, was significantly higher in the Cr-exposed workers than in the controls and it correlated with the blood Cr concentration. Although the concentration of MDA, the metabolite of lipid peroxidation, in the exposed workers was higher than that in the controls, no statistically significant correlation between the MDA level and the blood or urine Cr levels was observed. Accordingly, the genotoxicity and oxidative damage (plasma lipid peroxidation) in the Korean Cr-exposed workers were consequential at quite low exposure levels, plus chromosome rearrangement, especially translocation, was clearly evident as a biological response marker for Cr exposure based on a significant positive correlation between the translocations detected by FISH and the Cr in the blood.

Preferential reduction of dicentrics in reciprocal exchanges due to the combination of the size of broken chromosome segments by radiation.

Induction rates of the dicentrics and translocations involving chromosomes 2 and 4 in peripheral lymphocytes irradiated with X-rays at a dose of 3 Gy were examined using a conventional Giemsa staining method and a chromosome painting method. In total, 228 reciprocal exchanges detected in 982 metaphases were classified into three groups according to the break points of the original chromosomes. The incidence of both acentric fragments being larger than half of the original chromosome (combination 1) was only seven (3%) and did not contribute significantly to induction rates. When the broken acentric fragments of two affected chromosomes were smaller than half of the original chromosomes (combination 2), which was found in 175 (77%) rearrangements, the induction rates of dicentrics and translocations were about the same (86:89). But if the sizes of the broken segments were unequal in both chromosomes (combination 3: one with a larger acentric part and the other with a smaller acentric part), the yield of dicentrics was significantly lower than that of translocations (16:30). It was suggested that there was a special mechanism causing preferential reduction of dicentrics in reciprocal exchanges originated from the heteromorphic size of broken chromosomes in the last combination.

Chromosome analysis of Phyllodistomum folium (Trematoda, Gorgoderidae) infecting three European populations of zebra mussels.

The mitotic chromosome sets of the larval stages of Phyllodistomum folium infecting three European populations of Dreissena polymorpha were studied using conventional Giemsa staining and karyometrical analysis. The karyotype, described herein for the first time, consisted of nine chromosome pairs (2 n=18). The chromosomes were comparatively small and measured from 1.19 micro m to 5.22 micro m. The first and second longest chromosome pairs were, respectively, approximately 20% and 18% of the mean total chromosome set length and were clearly differentiated from the other seven pairs, which gradually decreased in size. Most chromosomes were biarmed with medially or submedially located centromeres; only pairs 1 and 4 contained subterminally located centromeres. No significant differences in the relative length values of corresponding chromosomes were observed between the three populations studied. The main interpopulation differences were detected in the centromeric index values of some corresponding chromosome pairs. These data, when analysed in conjunction with those of other Gorgoderidae, indicate that the karyotype of P. folium and other species in this family do not show any clear affinities with "typical" plagiorchiate chromosome sets.

Karyotypic Variation in the Genus Megaelosia (Anura, Hylodinae) with the First Description of a B-Chromosome in a Leptodactylid Frog

Three species of the genus Megaelosia (Megaelosia massarti, Megaelosia boticariana, and Megaelosia lutzae) were studied karyotypically using conventional Giemsa staining, C-banding, and NOR techniques. Megaelosia lutzae had 2n = 32 chromosomes, a new diploid number for this genus and for the subfamily Hylodinae. Two morphotypes of B-chromosomes were found in two specimens of M. massarti. C-banding patterns and NOR location varied among the species. Nevertheless, some chromosome pairs, as well as the NOR-bearing secondary constriction and the large pericentromeric C-block on the short arm of chromosome 10, were consistent among the three species. Megaelosia differs markedly from other Hylodinae and Dendrobatidae in terms of karyotype.

Comparative cytogenetic analysis of the South American alligators Caiman latirostris and Caiman yacare (Reptilia, Alligatoridae) from Argentina

Abstract One of the most important cytogenetic characteristics among the crocodilians is the diploid number, that ranges from 30 to 42 chromosomes and the fundamental number (FN) from 56 to 62, without sex chromosomes heteromorphisms. The purpose of this study was to characterise the chromosome complement of Caiman yacare and C. latirostris from Argentina through conventional Giemsa staining and simple banding techniques (CBC and NOR). The chromosome preparations were made from peripherical blood culture, and C- and NOR- banding were applied following routinary methods. The data obtained point out that both species share a similar chromosome number (2n = 42) and morphology with the complements composed by 12 pairs of telocentric chromosomes, 7 pairs of biarmed chromosomes and 2 pairs which could be considered microchromosomes. No conspicuous differences were revealed in the C-banding patterns and in both species the NOR’s was found on one of the small chromosome pairs.

Comparative cytogenetic studies of Bufo ictericus, B. paracnemis (Amphibia, Anura) and an intermediate form in sympatry

Specimens of Bufo ictericus, Bufo paracnemis and a third type, considered an intermediate subgroup between these species, were cytogenetically studied by conventional Giemsa staining, C-banding and staining of the nucleolus organizer region (NOR). The nuclear DNA content and seroproteins were also analyzed to characterize these species, and verify the possibility of hybridization between them. Karyotypes and cytogenetic markers were essentially equal on the basis of the methods used. The DNA nuclear content found was 6.25 ± 0.30 pg/DNA in Bufo ictericus; 7.57 ± 0.40 pg/DNA in Bufo paracnemis and 7.04 ± 0.29 pg/DNA in the intermediate subgroup. Eletrophoresis of total blood serum in Bufo ictericus, Bufo paracnemis and the intermediate specimens revealed a remarkable difference in the patterns of the protein bands whose molecular weight corresponded to that of albumin. While the parental species presented two different bands, the intermediate form presented 4. However, only three of these bands were seen in each specimen. The results obtained pointed to a high probability for natural hybridization between Bufo ictericus and Bufo paracnemis in the site and specimens studied.

An unusual karyotype with low chromosome number in Megacephalini, a basal group of tiger beetles (Coleoptera, Cicindelidae): cytogenetic characterisation by C-banding and location of rDNA genes

Mitotic and meiotic chromosomes of Megacephala brasiliensis, a representative of the tribe Megacephalini-a basal group in the family Cicindelidae-were studied using conventional Giemsa-staining, C-banding and fluorescence in-situ hybridization (FISH) with 18S rDNA probe. The diploid chromosome number was 2n=12 and the karyotype contained five pairs of metacentric and submetacentric autosomes and one pair of sex chromosomes, XY and XX in male and female, respectively. The X chromosome was metacentric while the Y was subtelocentric. All autosomes and the X showed distinct, large C-positive blocks around the centromere indicating extensive distribution of constitutive heterochromatin. The Y chromosome showed a conspicuous large C-positive block in the subterminal region as well as a weak interstitial band. FISH with a rDNA probe gave consistently positive signals in the pericentromeric region of the fourth pair of chromosomes. Size polymorphism of this rDNA-positive site was always observed. The karyotype of this beetle exhibits the smallest 2n known for any tiger beetle of the genus Megacephala and of other basal groups of the family though it shares single sex chromosome system with them. The extreme reduction in the autosome number appears to be the result of massive and combined rearrangenments of various types. The discovered case might parallel the famous “Muntjac scandal” in mammal cytogenetics.