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PubMed2006

Clinical diagnosis of cutaneous leishmaniasis: a comparison study between standardized graded direct microscopy and ITS1-PCR of Giemsa-stained smears.

Parasitological diagnosis of cutaneous leishmaniasis is absolutely necessary before treatment. Direct microscopy of scrapings taken from the margins of skin lesions is the most commonly used method for clinical diagnosis of leishmaniasis. In this study to evaluate the usage of stained smears as samples for PCR and the possible advantage of PCR, we compared the sensitivity of the diagnosis of Giemsa-stained skin scrapings by standardized graded direct microscopy with that of ITS1-PCR with the material of the same area of the slide. Three 5mm x 5mm squares were marked on each of the 20 Giemsa-stained touch smears from 20 clinically diagnosed Palestinian patients. Out of the 60 squares scanned for amastigotes under 100x oil-immersion light microscopy, 45 (75%) gave usable results and 23 of these were positive for Leishmania. Fifteen (25%) squares could not be scanned microscopically, 12 because of staining that was too thick and 3 because of inadequate staining. DNA from each scanned square was extracted separately after microscopy and run through ITS1-PCR. Of the 23 microscopy-positive squares, 20 (87%) of these were positive by PCR. Of the three that were negative, one failed to extract for DNA, the second showed only one amastigote in the entire square, and the third was normally graded as +1 but was not amplified for unknown reasons. Of the 22 squares negative for microscopy, 18 (82%) were ITS1-PCR positive. Additionally, all three improperly stained squares were ITS1-PCR positive. Of the 12 darkly stained squares, 11 were positive. A negative control group of 15 German individuals from which Giemsa-stained slides containing three squares each was prepared and these slides were also microscopically scanned and tested by ITS1-PCR. Both tests were negative with both methods. Compared to microscopy (data in parenthesis), PCR showed a sensitivity of 87% (37%) and a specificity of 100% (100%). We have concluded that Giemsa-stained smears are a readily usable sampling method for PCR and that ITS1-PCR is far more sensitive than microscopy.

Adolescent↗
PubMed1998

Immunohistochemical detection of parasite antigens in Theileria parva-infected bovine lymphocytes.

Theileria parva is the causal agent of East Coast fever (ECF), a fatal disease of cattle characterized by pyrexia, transient lymphadenopathy and panleukopenia. We have evaluated monoclonal antibodies (mAb) against three distinct antigens (p67, PIM and p32) of the parasite as immunohistological reagents for monitoring the kinetics of infection in cattle. Bovine lymphocytes were stained with the mAb at various intervals after infection in vitro and in vivo. The p67 sporozoite surface antigen was detected in only a small percentage of both, in vitro and in vivo infected cells. In contrast, expression of the polymorphic immunodominant molecule (PIM) of the parasite proved a useful indicator of infection and staining was correlated with the results of Giemsa analysis. PIM was detected from day 3 in in vitro-infected cells, but was not detected until day 5 in vivo after challenge with a 70% lethal dose of stabilized sporozoite. The p32 antigen was expressed only late in infection in vivo and its expression was associated with the development of merozoites. Less than 20% of in vitro-infected cells expressed p32. The immunohistochemical staining with anti-PIM mAb was found to be a useful tool for analysis of T. parva infection kinetics in cattle.

Animals↗
PubMed2000

Determination of optimal conditions for the immobilization of cells in a cell capture enzyme immunoassay (CC-EIA) by a simple Geimsa assay.

To determine the optimal conditions for the immobilization of cells in a cell capture enzyme immunoassay (CC-EIA), the most suitable diluent, and the optimal pH, temperature and period of incubation were examined using WI-38, a human embryonic lung fibroblast cell line. For the evaluation, we devised a simple Giemsa assay method, in which immobilized cells on a microplate were stained with Giemsa solution, the stained dye was eluted with ethanol after washing the plate, and the optimal density (O.D.) was measured at wavelength 620 nm. The optimal conditions for the immobilization were determined to be treatment with 5% formalin in phosphate-buffered saline (PBS) (pH 7.2) for 15 minutes at room temperature, which were confirmed to be suitable for the measurement of cell associated collagen by CC-EIA. Additionally, we found that the simple Giemsa staining method was also useful for evaluating the number of immobilized cells on the microplate after CC-EIA.

Azure Stains↗
PubMed1987

Histological techniques for the study of granular cells during wound healing.

We have used GMA-embedded tissues sectioned 2-3 mu for the identification of granulated cells in wounded and normal macaque tissues. Mast cell granules stain brilliantly with Giemsa or with PAS counterstained with alcian blue, neutrophils stand out clearly when treated with the PAS method and macrophage granules stain with PAS as well as with basic dyes. These techniques are helpful for the identification of these cells during wound healing and during inflammatory processes.

Animals↗
PubMedDate not supplied

Cytologic detection of microsporidia spores in bile. A comparison of stains.

OBJECTIVE: To compare stains in preparations of bile in a patient with AIDS and microsporidial cholangitis. STUDY DESIGN: Bile was obtained from a 30-year-old male with AIDS and symptoms of cholangitis. Comparative staining of the specimen was performed using a formalin-fixed preparation stained with Chromotrope 2R stain and with alcohol-fixed preparations stained with Gram and Giemsa stain and Diff-Quik. An alcohol-fixed ThinPrep slide was stained with Papanicolaou stain. RESULTS: Diagnostic microsporidia spores were detected under oil immersion using Papanicolaou, Chromotrope 2R, Giemsa and Gram stain. The Diff-Quik-stained preparation also revealed microsporidia but with suboptimal morphology. CONCLUSION: Detection of microsporidia in bile can be achieved using several different stains routinely available to cytologists, most optimally with alcohol-fixed Papanicolaou- or Giemsa-stained preparations or with Chromotrope 2R stain, which is available in parasitology laboratories. These findings should be applicable to fluids from other body sites with this emerging pathogen in AIDS.

Adult↗
PubMed2003

Evaluation of the direct acridine orange staining method and Q.B.C. test for diagnosis of malaria in Delhi, India.

Conventional Giemsa stained peripheral blood smear examination for demonstration of malarial parasites remains the gold standard for diagnosis of malaria in developing endemic countries. However this technique is time consuming, requires training and may give poor results in cases with low parasitaemia. To overcome these problems and improve diagnostic accuracy two newer tests have been studied and compared with standard Giemsa staining. These are the wet mount fluorescence microscopy of Acridine Orange stained thin blood films (A.O.) and the Quantitative Buffy Coat technique (Q.B.C) for diagnosis of malaria. A.O. staining was found to be 97.5% sensitive and 100% specific for detection of all stages and species of malarial parasite. The Q.B.C assay was found to be 100% sensitive and 97.5% specific for diagnosis of malaria. A.O. staining was very fast and the species identification was easy once the staining was optimised. The Q.B.C. test required considerable amount of practice, costly equipment, however it was fast and in our study was found to be highly sensitive.

Acridine Orange↗
PubMed1980

Analysis of crossing over in mouse meiotic cells by BrdU labelling technique.

Differential staining of sister chromatids in male mouse meiotic cells was achieved by continuous infusion with BrdU from the tail vein followed by Giemsa staining (the FPG technique of Perry and Wolff, 1974). Analysis of 341 bivalents including XY, and 21 univalents reveals that: (1) visible crossing over coincided exactly with the site of chiasmata; (2) no evidence was obtained in support of chiasma terminalisation; (3) an anomalous type of crossing over was found in the monochiasmatic bivalents, which could not be explained by the conventional hypothesis for crossing over; (4) some of the terminally associated bivalents might be achiasmatic, and univalents might have originated from such bivalents; (5) in XY bivalents, sister chromatid association was between lightly and darkly stained chromatids, suggesting a lack of crossing over and the existence of other genetic controls for this association; (6) during meiosis sister chromatid exchanges (SCEs) might be less frequent than crossovers.

Animals↗
PubMed1989

Comparison of methenamine silver nitrate and Giemsa stain for detection of Pneumocystis carinii in bronchoalveolar lavage specimens from HIV infected patients.

Seventy-seven bronchoalveolar lavages from human immunosuppressive virus infected patients with pulmonary symptoms were examined routinely for the presence of Pneumocystis carinii, using Giemsa stain to detect trophozoites and methenamine silver nitrate to detect cysts. Thirty-seven samples were found positive with both methods, but a further 10 were diagnosed with Giemsa indicating that the trophozoite stain is more sensitive. As Giemsa stain is simple, quick, cheap and familiar to most microbiological laboratories it should be used for screening of samples to be examined for Pneumocystis carinii. In cases where trophozoites are not detected by Giemsa a supplementary cyst stain may be performed in order to rule out the presumably few cases where cysts are present as the only evidence of Pneumocystis carinii infection.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions.

OBJECTIVE: To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. STUDY DESIGN: Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. RESULTS: Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. CONCLUSION: Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.

Azure Stains↗
PubMed2003

Utility of thick smears of bone marrow aspirate in pyrexia of unknown origin.

OBJECTIVE: To determine the diagnostic efficacy of Giemsa stained thick smear of bone marrow aspirate for evidence of malaria in febrile individuals in whom the diagnosis was otherwise unknown. DESIGN: Descriptive case-series study. PLACE AND DURATION OF STUDY: This study was conducted at the Department of Medicine, Sandeman Provincial Hospital, Quetta, in the Balochistan province of Pakistan, between January 1994 and December 1998. SUBJECTS AND METHODS: A total of 80 patients were included in the study, who presented with history of fever for more than 3 weeks with splenomegaly and pancytopenia. The bone marrow specimens of all these patients were examined. The bone marrow was aspirated from the posterior superior iliac spine. Three sets of smears were made. One set of thin smears was stained by Leishman's stain after air-drying and the second set of thin smears was air-dried, fixed with 97% methanol and later stained with 5% Giemsa stain. The third set of smears was a thick smear stained unfixed with 5% Giemsa stain. A temperature of 37 degrees C and a pH of 7.2 was maintained during the staining process. The presence of malarial parasite in the bone marrow was determined. Besides, the degree of megaloblastic change in erythroid series and the bone marrow cellularity were evaluated. The results obtained were recorded and statistically analyzed. RESULTS: Of the 80 bone marrows examined, 32(40%) showed the presence of malarial parasites, 19(23.75%) showed haematological malignancies, 16(20%) showed bone marrow suppression while 12(15%) showed megaloblastic changes. One (1.25%) case showed Leishman-Donovan bodies. Out of 32 cases showing the presence of malarial parasite in bone marrow, only 5(15.6%) revealed parasites, i.e. Plasmodium falciparum gametocytes on thin smear while 32(100%) cases showed Plasmodium falciparum trophozoites and gametocytes on thick smear. The ratio was 1:6.4. These 32 patients were divided into two groups, one group having mild anaemia (>8 g/dL) and the other severe anaemia (Hb<8 g/dL). In this study 20 patients had mild and 12 had severe anaemia. Megaloblastic changes were seen in 15 cases. Hypocellularity was seen in 3 patients with falciparum malaria while 28 showed normocellularity. The marrow was hypercellular in one patient. CONCLUSION: In patients presenting with prolonged fever, splenomegaly and pancytopenia, the Giemsa stained thick smear of bone marrow aspirates for evidence of malarial parasites was far superior to thin smear in diagnosing Plasmodium falciparum malaria.

Adult↗
PubMed2006

Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years.

This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-stained slides can provide a Leishmania DNA source for performing clinical and epidemiological studies of leishmaniasis.

Animals↗
PubMed1988

Studies on the chromosome by scanning electron microscope--a new trial with long-term Giemsa staining.

Chromosomes, which can be seen under a light microscope (LM) as bands, are too thin to see with a scanning electron microscope (SEM). We noticed that chromosomes increase in thickness with Giemsa staining, so that scanning microscopic specimens could be prepared by long-time Giemsa staining for SEM observation, and in the present study the following results were obtained. 1) When the duration of the trypsin treatment was kept constant, and the Giemsa staining was performed from 30-min to 24-h, it was found that the thickness of the chromosomes increased with the length of treatment time, and the fibrous structures were clearly seen. 2) While a sufficient thickness of the chromosomes was obtained with the long-time Giemsa staining, the bands became unclear as the time of staining was extended. 3) A long-time Giemsa staining without trypsin treatment showed more "highly packed" fibers, so that the trypsin treatment was felt to be related to the dispersion of the fibers.

Azure Stains↗
PubMedDate not supplied

Comparative effectiveness of light-microscopic techniques and PCR in detecting Thelohania solenopsae (Microsporidia) infections in red imported fire ants (Solenopsis invicta).

The main goal of this study was to compare the effectiveness of three staining techniques (calcofluor white M2R, Giemsa and modified trichrome), and the polymerase chain reaction (PCR) in detecting the microsporidium Thelohania solenopsae in red imported fire ants (Solenopsis invicta). The effect of the number of ants in a sample on the sensitivity of the staining techniques and the PCR, and the effect of three DNA extraction protocols on the sensitivity of PCR were also examined. In the first protocol, the ants were macerated and the crude homogenate was used immediately in the PCR. In the second protocol, the homogenate was placed on a special membrane (FTA card) that traps DNA, which is subsequently used in the PCR. In the third protocol, the DNA was purified from the homogenate by traditional phenol-chloroform extraction. Except for PCR using FTA cards, the sensitivity (number of samples positive for T. solenopsae) of all detection techniques increased with the number of ants in the sample. Overall, Giemsa was the least sensitive of all detection techniques. Calcofluor was more sensitive than modified trichrome with ants from one site and was equally as sensitive as PCR with crude DNA or a FTA card with ants from both sites. Trichrome staining was equally as sensitive as PCR with a FTA card at both sites, but it was less sensitive than PCR with crude DNA at one site. PCR on FTA cards was less sensitive than PCR with crude DNA for ants from one site but not the other. There was no difference whether crude or phenol-chloroform purified DNA was used as template. In summary, the results of this study show that PCR based on a crude DNA solution is equal to or more sensitive in detecting T. solenopsae than the other detection techniques investigated, and that it can be used as a reliable diagnostic tool for screening field samples of S. invicta for T. solenopsae. Nevertheless, ant smear stained with calcofluor or modified trichrome should be used to buttress findings from PCR.

Animals↗
PubMed1979

Identification of the facultative heterochromatic X chromosome in females of 25 rodent species.

Treatment of the chromosomes of 25 rodent species with a 50 degrees C hypotonic solution and Giemsa staining permitted identification of the heterochromatic X chromosome in 24 species. With this technique, the facultative of the heterochromatic X chromosome or the facultative portion of large, composite-type X chromosoms is stained darker than the other chromosomes, allowing it to be distinguished from the homologous euchromatic X chromosome in female metaphase cells. Intense staining of the single X chromosome was not observed in male metaphase cells. It is suggested that this differential staining of one of the two X chromosomes might be due to qualitative differences in chromosomal proteins rather than to differences in the degree of chromosomal condensation or in DNA base sequence.

Animals↗
PubMed2001

Detection of stable chromosome aberrations by FISH in A-bomb survivors: comparison with previous solid Giemsa staining data on the same 230 individuals.

PURPOSE: To evaluate the relative abilities of the solid Giemsa staining (conventional) and fluorescence in situ hybridization (FISH) methods in the detection of stable chromosome aberrations in the peripheral blood lymphocytes of A-bomb survivors. MATERIALS AND METHODS: Lymphocytes from a total of 230 A-bomb survivors for whom prior chromosome aberration data had been obtained by the conventional method were recently examined afresh using FISH in which chromosomes 1, 2 and 4 were painted with composite probes. RESULTS: It was found that the early use of the solid Giemsa staining method had allowed the detection of translocations with a mean frequency of 73% of the value for the genome-equivalent translocation frequency (F(G)) that was now obtained using FISH. The disparity may at least in part be due to the reciprocal exchange of seemingly identical amount of chromosome material; such exchanges can escape detection by the conventional method but can be readily identified using FISH. CONCLUSION: It has previously been established that the conventional method can detect about 20% of radiation-induced translocations as abnormal monocentric chromosomes. Present results indicate that an additional 50% can be detected if proper karyotyping is conducted and the remaining 30% are not likely to be detected unless FISH or banding methods are used. Thus, solid Giemsa staining accompanied by karyotyping may not be quite as unsuitable as is generally assumed for retrospective biodosimetry analyses, which deal mainly with stable aberrations.

Azure Stains↗