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PubMed1983

Staining air dried protoplasts for study of plant chromosomes.

The air drying technique used in mammalian cytology was applied to isolated plant protoplasts for study of chromosomes. For cultured celery cells, this technique resulted in good spreads of metaphase chromosomes with high resolution. Mitotic chromosomes of Brassica species are relatively small, poor stained by common stains, and difficult to spread by the squash technique. In this study, however, the chromosomes of B. carinata in callus culture were spread well and stained clearly with Giemsa staining solution. The chromosome preparations by the present techniques should also be amenable to chromosome banding studies in plants.

Azure Stains↗
PubMed1994

Giemsa as a fluorescent stain for mineralized bone.

We present evidence for a previously unrecognized differential staining effect of Giemsa solution in fluorescence microscopy. The effect consists of selective fluorescent staining of mineralized bone (and elastic fibers) in tissue sections and, like the classical Romanowsky effect, is based on the differential binding of Eosin Y to tissue structures in the presence of Azur II and Methylene Blue. This effect opens the way to new applications of the Giemsa solution in fluorescence microscopy and in confocal fluorescence microscopy.

Animals↗
PubMed1986

Cytophotometric analysis of neuronal chromatin and RNA changes in oxotremorine-treated rats.

Neuronal nucleic acid responses were examined within the rat striatum and sensorimotor cortex (layer V) following single intraperitoneal injections of the central cholinergic-muscarinic agonist oxotremorine (0.1, 0.7, or 1.0 mg/kg). After stoichiometric Feulgen and azure B staining of brain sections, scanning-integrating microdensitometry was used to quantify Feulgen-deoxyribonucleic acid levels, changes in the susceptibility of chromatin to Feulgen acid hydrolysis (F-DNA yield) and azure B-ribonucleic acid (RNA) content of neurons on an individual basis. Changes in neuronal nuclear and nucleolar volumes were also determined histometrically. Within the striatum and sensorimotor cortex, oxotremorine produced marked dose-dependent elevations in both F-DNA yield and RNA content. These metabolic increases were typically paralleled by elevations in nuclear and nucleolar volumes. The data demonstrate that the oxotremorine-induced central muscarinic activation is associated with dose-related enhancements in neuronal chromatin template activity, RNA content, and protein synthetic capacity.

Animals↗
PubMed2002

The wright-giemsa stain. Secrets revealed.

The colorful story of the development of the Wright-Giemsa stain is retold. Dramas are replayed, secrets are exposed, and laurels are properly returned to scientists long forgotten. The delicately balanced chemical composition of the stain, once enigmatic, is defined. Finally, an attempt is made to unshroud some of the mystery surrounding the staining method; tried-and-true procedures and useful snippets of laboratory lore are provided. Scientific explanations and reliable methods aside, however, the stain continues to possess a certain mystique, seemingly consisting of equal parts technical expertise, art, and magic.

Azure Stains↗
PubMed1994

Preservation of cytoplasm in the human lymphocyte micronucleus assay.

A major limitation in the quantitative accuracy of the human lymphocyte micronucleus (MN) assay is preservation of the cytoplasm during the cell harvesting. In this short communication, an improved method for cytoplasm preservation in a cytokinesis-blocked, whole-blood microculture (0.3 ml) technique is described. We believe that the timing of the hypotonic treatment, speed of centrifugation, handling of the cell suspension and proper Giemsa staining are important variables in the human peripheral lymphocyte MN assay.

Azure Stains↗
PubMed1987

Microwave-stimulated staining of plastic embedded bone marrow sections with the Romanowsky-Giemsa stain: improved staining patterns.

Staining plastic sections with the Romanowsky-Giemsa method is both time-consuming and difficult. This paper reports how the staining time can be reduced to 25 min using microwave irradiation of the staining solution. It is shown that staining results depend on the fixative used, staining temperature, dye concentration and pH of the staining solution as well as on several parameters of the microwave irradiation technique. The staining patterns are improved when compared with those obtained by conventional staining of plastic sections. The colors are more brilliant and greater contrasts are observed. Basophilia, polychromasia, and orthochromasia accompanying red cell maturation are more pronounced. For white cell maturation the initial appearance of specific granules (neutrophil, basophil, and eosinophil) is more evident. Thus, cell classification is easily accomplished using the described technique. It is suggested that microwave-stimulated staining be considered for routine use.

Animals↗
PubMed1997

Analysis of malaria parasite RNA from decade-old Giemsa-stained blood smears and dried mosquitoes.

We have analyzed RNA isolated from recently prepared and historically preserved slides containing smears of Plasmodium-infected blood. We found that slides preserved as long as 20 years can yield RNA that is a suitable template for polymerase chain reaction (PCR) amplification. Mosquitoes that have been stored for years under ambient temperature can also be used as an RNA source. The RNA amplification from slide-derived material is shown to be dependent upon the addition of reverse transcriptase and the resultant products are specific to the developmental state of the parasite. Amplification of ribosomal RNA with primers conserved for Plasmodium and hybridization with species-specific probes provide a general, unbiased method for species determination. Messenger RNA transcripts from slides also appear to serve as templates. The procedure may add complementary information to that derived from microscopic examination of Giemsa-stained blood smears including species identification, variant antigen identification and drug resistance status.

Animals↗
PubMedDate not supplied

[Microscopic diagnosis of Pneumocystis carinii. Comparison of various staining technics].

The toluidine blue 0 and methenamine-silver nitrate staining techniques allowed a good microscopic distinction of Pneumocystis carinii cysts in lung autoptic impression smears. Poor results we observed with methylene blue and modified Gram techniques. With Giemsa's stain the intracystic bodies are shown, however the microscopic examination requires experience and patience.

Azure Stains↗
PubMed1980

Effects of acetic acid-alcohol, trypsin, histone 1 and histone fragments on Giemsa staining patterns in chromosomes.

In an effort to minimize subjective bias, a classification scheme was devised to assess Giemsa staining patterns obtained with experiments involving acetic acid-alcohol and exogenously applied histone 1 and polypeptides. A single rinse of metaphase preparations with acetic acid-alcohol quantitatively reduced Giemsa dye binding. Acid-alcohol irreversibly changed the conformation of H1 and its ability to interfere with trypsin G-banding. Our results suggest that, in addition to protein extraction, acid-alcohol may alter the conformation of acid-insoluble components of metaphase chromosomes. The carboxy-terminal polypeptide (residues 73--212) from NBS cleavage of H1 was an effective inhibitor of Giemsa staining and trypsin G-banding. However, this polypeptide which is preferential for supercoiled DNA was much less efficient in inhibiting Giemsa staining of trypsinized metaphase chromosomes. The molecular consequences of these experiments are discussed.

Acetates↗
PubMed1985

Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease.

A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.

Animals↗
PubMedDate not supplied

Comparative mouse micronucleus evaluation in bone marrow and spleen using immunofluorescence and Wright's Giemsa.

Bone marrow and spleen toxicity, clastogenicity and aneugenicity were analyzed in the CD1 mouse using an antikinetochore antibody (AKA) procedure (Krishna et al., Mutation Res., 282, 159-169, 1992). Further, to verify the fluorescence micronucleus (MN) analysis, additional slides were stained with Wright's Giemsa and results were compared. 5 mice per sex were treated with cyclophosphamide (CP) (40 mg/kg) or vincristine (VC) (0.1 or 0.2 mg/kg). Slides were prepared 24 h postdose using a column fractionation procedure. Per animal, 400 total erythrocytes (TEs) for toxicity and 2000 polychromatic erythrocytes (PCEs) for MN per tissue were analyzed. In the fluorescent method, the clastogen, CP, produced MNPCEs predominantly devoid of kinetochores (K) and the aneugen, VC, produced mostly MNPCEs containing K. The MNPCE frequency did not differ significantly between tissues; however, it differed statistically between sexes. On an overall basis, spleen had significantly lower PCE to TE ratios compared to bone marrow. In general, CP and VC caused a small, but statistically significant decrease in PCE frequencies compared to controls, suggesting possible toxicity to these tissues at the given doses. The data on Wright's stain indicated that the proportion of PCEs and MNPCEs in general, were comparable to those using fluorescent stain. This study further confirms the usefulness of an AKA-staining technique in a multiple genetic endpoint evaluation under a single set of microscopic conditions.

Animals↗
PubMed1980

Sequential staining of euchromatic and heterochromatic regions of the human Y chromosome.

A sequential silver-Giemsa (SG) procedure is presented, initially to stain the p11 and q11 euchromatic bands and subsequently the q12 heterochromatic band of the human Y chromosomes. A three sub-band division of the q11 band can be identified. The same technique differentially stains the secondary constriction of chromosome 9 as well as most other satellite III DNA regions of the human karyotype.

Azure Stains↗
PubMedDate not supplied

Modified Giemsa staining for rapid diagnosis of malaria infection.

OBJECTIVES: To develop and evaluate a rapid method for the diagnosis of malaria infection by microscopy of stained blood films. SUBJECTS AND METHODS: Blood specimens were collected from randomly selected confirmed malaria cases (n = 75) and suspected malaria cases (n = 175). The microscopy was done on each set of blood films stained by modified and the standard Giemsa staining methods. RESULTS: All the 75 previously diagnosed malaria cases were confirmed by the microscopy of blood films stained by both methods. Forty-nine (28%) of the 175 cases suspected for malaria infection showed malarial parasites on microscopy of blood films stained by both methods. However, due to homogeneous staining and clearer background of the blood films it was possible to determine the parasite species in 65% of the cases on microscopy of the thick films stained with the modified method compared to only 20% with the standard method. Further, the turnaround time for reporting the microscopy test result was 15-20 and 45-50 min with modified and standard staining methods, respectively. CONCLUSION: Our data showed that performance of the modified staining method in detecting malarial parasites was comparable to that of the standard staining method. Moreover, the modified staining method was rapid, easy to use, and reliable.

Animals↗
PubMed1995

Comparison of acridine orange and giemsa stains for malaria diagnosis.

Recently, the Acridine orange (AO) staining method has improved for identification of malaria parasites. Fixed and preserved blood smears of malaria patients were used for comparative analysis of AO and Giemsa stains. The AO staining method required less time and was more sensitive under lower magnification than the Giemsa staining method. The AO staining method provides an alternative to Giemsa for malaria diagnosis in the field and laboratory.

Acridine Orange↗
PubMed1979

Cytological analyses of 14p+ variant by means of N-banding and combinations of silver staining and chromosome bandings.

An inherited human karyological variant (14p+) has been studied with a number of cytochemical techniques. The short arm of this variant chromosome 14 is nearly as long as the long arm, giving the chromosome a submetacentric to metacentric appearance. In conventionally Giemsa-stained preparations, maximum of three secondary constrictions can be observed the marker arm. The secondary constrictions are silver-positive in Ag-NOR preparations. However, the entire arm stains deeply in N-banded preparations. The 14p+ arm is also Q-negative, C-negative, G-negative, and R-positive with an almost homogeneous texture. The difference between N-banding and silver staining is interpreted as the result of gene activities of the ribosomal cistrons.

Azure Stains↗
PubMed1981

Structural organization of the heterochromatic region of human chromosome 9.

Giemsa-11, G-banding and Lateral Asymmetry staining techniques were used to define the substructure of the C-band heterochromatin of human chromosome 9, in a sample of 108 different chromosomes 9, from 54 individuals. In this sample, the juxtacentromeric portion of the C-band region stained positive by the G-banding technique while Giemsa-11 delineated a more distally located block. Examination of the pericentric inversions generally revealed that the entire C-band region is changed with the substructural organization left intact; i.e. the G-band is proximal, the G-11 distal to the centromere. The "partial pericentric inversions" were found to have larger than average amounts of G-band heterochromatin on the short arm. The G-11 staining was in its usual position on the long arm with none on the short arm. Such apparent inversions therefore may not represent true inversions. Long heterochromatic regions frequently had a segmented appearance when stained with G-11; there was a dark G-band within the pale heterochromatic region when stained with the G-banding technique which corresponded in location to the achromatic gap produced by G-11. This extra G-band may have been derived from the juxtacentromeric G-band by processes analogous to unequal crossing over. Simple lateral asymmetry was consistently present only in the G-band heterochromatin of those chromosomes 9 containing large blocks of G-band positive material. Examination of the portion of the C-band which would correspond to the G-11 positive material revealed no consistent patterns of asymmetry. Usually both strands were heavily stained and symmetrical but occasionally there were light areas present on one strand suggestive of compound lateral asymmetry.

Azure Stains↗
PubMed2000

Bleomycin-induced chromosome aberrations in head and neck cancer patients analyzed by classical cytogenetics and FISH.

Individual sensitivity to mutagens has been considered to play an important role in head-and-neck squamous cells carcinoma (HNSCC) development. The bleomycin test was introduced for establishing constitutional susceptibility to mutagens (T.C. Hsu, D.A. Johnston, L.M. Cherry, D. Ramkisson, S.P. Schantz, J.M. Jessup, R.J. Winn, L. Shirley, C. Furlong, Sensitivity to genotoxic effects of bleomycin in humans: possible relationship to environmental carcinogenesis, Int. J. Cancer 43 (1989) 403-409). Its criteria are based on scoring of chromosome aberrations (CAs, mainly breaks) in Giemsa-stained chromosomes. Fluorescence in situ hybridization (FISH) offers an easy method for analysis of translocations, acentric fragments and dicentrics. In the present study FISH was applied in the analysis of bleomycin-induced CAs of the HNSCC patients and controls. The results proved that FISH is a complementary method to the classical staining in monitoring of bleomycin-induced CAs.

Adult↗