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Several different methods for identifying human chromosomes were developed over the past years. These methods include fluorescence techniques using quinacrine mustard, acridine orange or Hoechst 33 258 and special methods using Giemsa staining. Studies on replication of chromosomes were greatly improved by using BUdR labelling.
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A DNA body is present in the females of the fly Tipula oleracea and is formed in contact with the sex chromosomes in the oogonial interphases. At each oogonial mitosis, the DNA body follows the chromosomes to one anaphase group and is included in one of the telophase nuclei. The body increases appreciably in size during the interphase of meiosis. All oocytes have the body, but only a few nurse cells possess it. The DNA body synthesizes its DNA at a different time than the chromosomes, as is shown by incorporation of tritiated thymidine, and contains 59% of the DNA of the nucleus, as is disclosed by spectrophotometric measurements. At late diplotene the DNA body disintegrates, releasing its DNA into either the nucleus or the cytoplasm. When studied in the electron microscope, the DNA body appears composed of a tight mass of intertwined fibrils. Demonstration that the main mass of the body is composed of DNA is obtained from cytochemical tests which reveal that the DNA body is Feulgen positive, stains green with azure B, incorporates H(3)-thymidine, and after digestion with DNase is Feulgen negative. The DNA of the body is complexed with histone, like the DNA of the chromosomes, as is revealed by an intense alkaline fast green staining. Electron microscope examination of oocytes reveals that one side of the DNA body is in close contact with the nuclear envelope and that the other side possesses an outer shell composed mainly of particles 150 to 250 A in diameter. Between the outer shell and the chromosomes there is a band of low electron opacity, 4000 to 7000 A thick. In the light microscope, this light band together with the outer shell is Feulgen negative and stains violet with azure B; this is confirmation of the presence of RNA. In the oocytes the nucleoli are found inside the DNA body. These nucleoli have a nucleolonema composed mainly of particles 150 to 250 A. The nucleoli are Feulgen negative, alkaline fast green negative, stain violet with azure B, and do not stain with azure B after RNase digestion, thus confirming their RNA content. The presence of the nucleoli inside the DNA body and of a band of RNA between the body and the chromosomes is indicative of a high RNA synthetic activity. Since the DNA of the body is complexed with histone, as in the chromosomes, and the nucleoli are located inside the body, the simplest interpretation of the DNA body is that it represents hundreds of copies of the operons of the nucleolar organizing region or neighboring regions. The situation found in Tipula has several basic features in common with the polytene chromosomes of other Diptera and with the hundreds of nucleoli present in Triturus oocytes. In all three cases, genes seem to be copied hundreds of times but are kept in different types of packages. A DNA body like the one in Tipula oleracea is found in other species of Diptera and in the Coleoptera. There is no indication, from the present investigation, that the DNA body is in any way associated with a virus.
We compared the value of the Wright-Giemsa stain with the Papanicolaou stain for diagnosing hairy cell leukemia in the body cavity fluids of three patients. The cytological features of the leukemic cells were much clearer on the Wright-Giemsa-stained preparations. This allowed for the accurate diagnosis of leukemic involvement of the body cavity fluids in 3/3 patients using the Wright-Giemsa stain but only 1/3 using the Papanicolaou stain. A tartrate-resistant acid phosphatase stain was used to confirm the diagnosis in each case.
Morphology and staining characteristics of Ehrlichia bovis were observed after staining with Romanowsky and Gimenez stains and fluorochroming with acridine orange. Ehrlichia bovis could be identified as elementary bodies, initial bodies and morulae in the host cell cytoplasm. The inclusions were solid and compact and resembled more closely those of E. canis (canine monocytic ehrlichia) than E. phagocytophilia or E. ondiri (bovine granulocytic ehrlichiae). The organisms usually took acidophilic shades with Romanowsky stains. Apart from morulae, the other forms sometimes resembled azurophil granules. While Gimenez staining and fluorochroming by Anderson and Greiff technique gave poor results, the organism stained well on fluorochroming with Lauer's technique.
Malaria is a parasitic infection caused by Plasmodium species and it is especially seen in tropical and subtropical areas. Our aim was to determine whether or not malaria is transmitted by blood transfusion in Turkey and to define the rate and the differences between endemic and non-endemic areas. During this study, blood samples were taken from donors who applied to Blood Banks in Istanbul (non-endemic area) and in Adana (endemic area). 2229 donors were screened using the OptilMAL Rapid Malaria Test and Giemsa staining method. Neither the OptiMAL Rapid Malaria Test nor the gold standard Giemsa staining method detected infected erythrocytes and Plasmodium lactate dehydrogenase.
Giemsa-stained blood smears are mainly used for microscopic examination to diagnose malaria. However, they may also be subjected to polymerase chain reaction (PCR) to confirm diagnosis or for retrospective studies requiring the analysis of old smears. We investigated the possibility of DNA contamination occurring during automated Giemsa staining or due to the failure to clean the oil-immersion objective during microscopic examination. We tested blood smears from uninfected, Plasmodium vivax-infected, and P. falciparum-infected patients. DNA contamination was observed after both staining and microscopy, although contamination was unpredictable during staining. These results are of utmost importance when smears are used for PCR.
Treatment with weak solutions of 8M urea and staining with Giemsa produces banding in the euchromatin of Drosophila virilis somatic chromosomes.
To determine the volume of bronchoalveolar lavage (BAL) fluid necessary to diagnose Pneumocystis carinii pneumonia (PCP) in immunocompromised patients, specimens from 25 patients were evaluated. Twenty-one patients were HIV infected. BAL was performed using three to four 60-mL aliquots of room temperature, sterile, saline solution. Each syringe of BAL effluent was numbered and its volume was measured. Immunofluorescent stains were performed on about 8-mL aliquots of the initial, final, and aggregate BAL specimens, and a modified Giemsa stain was also performed on a 0.4-mL aliquot of the aggregate specimen. Of 25 patients, Pneumocystis carinii organisms were identified in 9 with HIV infection, in whom all BAL specimens were positive with both immunofluorescence and Giemsa stains. In 16 patients, BAL specimens were negative for P carinii on both immunofluorescent and modified Giemsa testing. Both staining methods were 100% specific (95% confidence interval [CI], 83 to 100%) and 100% sensitive (95% CI, 72 to 100%). The volume of BAL effluent in the initial specimens positive for P carinii ranged from 15 to 25 mL. We conclude that in this small group of patients, PCP was accurately diagnosed from a single 60-mL BAL specimen stained with immunofluorescence methods.
PURPOSE: To find the better method of eosinophilic granulocytes staining by comparison between HE staining and Giemsa staining METHODS: Twenty conjunctivitis patients(40 eyes) were studied. The infected palpabral conjunctiva of both eyes were scroped and stained separately with Giemsa staining and HE staining. RESULT: Under microscope, eosinophilic granulocytes were more clear by Giemsa staining than HE staining. CONCLUSION: In showing eosinophilic granulocytes, Giemsa staining was better than HE staining.
This paper deals with staining of DNA-phosphate groups with a mixture of an equal parts of aqueous solution of azure A and acridine yellow in a 1:1 proportion and also embodies a study of the absorption properties of the stained nuclei. It also embodies results of sequential staining of nuclei stained first with azure A followed by staining with acridine yellow and vice versa, after extraction of RNA with cold phosphoric acid. The results indicate that the absorption peaks of nuclei differ from those of nuclei stained for DNA-aldehyde molecules with azure A-SO2 or acridine yellow-SO2. The in vitro absorption characteristics of an aqueous solution of azure A and those of an aqueous solution of acridine yellow are also presented herein. The conclusion obtained from this study is that all the phosphate groups of DNA do not take part in the staining process when staining is carried out with azure A or acridine yellow alone after after RNA has been extracted. This is because the nuclei stained with these dyes sequentially show the presence of acridine yellow-DNA and azure A-DNA complex.
During routine evaluation of trypan blue-Giemsa stained semen smears, sperm cells can be found with unstained heads and with stained tails. It was hypothesized that these cells were immotile and should not be considered as live. Sperm motility was determined in isoosmotic, and presumably isotonic trypan blue-stained wet preparations. Bull, ram and boar semen smears were stained with hypoosmotic trypan blue-Giemsa to compare the relationship between the percentage of stained sperm tails and the percentage of sperm tails remaining straight under hypoosmotic conditions. Actively moving spermatozoa with unstained heads, but with stained tails were never observed in wet preparations. The correlation coefficient found between the percentage of sperm with stained tails and the percentage with straight tails was 0.81, 0.94 and 0.85 for bull, ram and boar spermatozoa, respectively. Results of this study show that sperm cells with an intact head membrane, but a stained and presumably membrane-damaged tail are not motile. Therefore these cells should be included in the dead category rather than alive in the usual live-dead studies with vital stains.
By eliminating the human evaluation variable, it was possible to carry out an investigation into the 'true' association between normal sperm morphology outcomes assessed according to the World Health Organization guidelines and strict criteria. Two computer-assisted semen analysis systems were used, IVOS and Mika, to evaluate Diff-Quik and Papanicolaou stained slides. As expected, the mean normal sperm morphology outcomes for the World Health Organization classification evaluations were markedly higher for both the Diff-Quik (mean difference = 40.13%) and the Papanicolaou (mean difference = 32.55%) stained slides. The association between the outcomes were low for the Diff-Quik stained slides (r = 0.379) and poor for the Papanicolaou stained slides (r = 0.110). While the association achieved with the computer-assisted semen analysis systems using Diff-Quik stained slides was comparable to the association between the manual evaluations (r = 0.386), the manual evaluation of Papanicolaou stained slides produced a relatively good association (r = 0.690). Although the numbers were small, the results show the probability of poor class correlations. Approximately 40% of outcomes were incorrectly classed at a 14% (strict criteria) and 50% (World Health Organization guidelines) cut-off point for both staining methods. This study confirms the fundamental differences between the two classification systems. The results also indicate that, of the two stains used, Diff-Quik should be the preferred staining method for computer-assisted sperm morphology evaluations.
Three methods of isolation and identification of Chlamydia trachomatis inclusions in cell monolayers were compared: Giemsa staining followed by darkfield microscopy of cycloheximide treated Buffalo green monkey kidney monolayers (BGM), fluorescent monoclonal antibody staining of cycloheximide treated BGM and of HeLa 229 monolayers. In an unselected group of 895 patients with suspected chlamydial infections including contacts of symptomatic patients, significantly more positive chlamydia isolates were detected with immunofluorescent monoclonal antibody staining of BGM and HeLa 229 than with Giemsa staining followed by darkfield microscopy on BGM. However, there was no disparity between the 2 staining methods with specimens from 156 patients with untreated non-gonococcal urethritis. In all chlamydia positive specimens, significantly more inclusions were seen after BGM-immunofluorescence than after HeLa-immunofluorescence or BGM-Giemsa. The extra cost associated with immunofluorescence staining was justified by the time saved in scanning.
Routine semen examination remains an important tool for the diagnosis and treatment in human subfertility. Of all semen parameters, sperm morphology seems to be one of the most powerful indicators of a man's fertilizing potential in vitro and in vivo. Lack of standardization of sperm morphology assessments remains the main reason for the usefulness of this parameter. The aim of this study was to analyze the agreement between the wet-stained preparations versus those stained with modified Diff-Quik for sperm morphology. A total of 100 unselected semen samples from infertile couples were analyzed. Sperm morphology was evaluated with unstained specimens and following modified Diff-Quik staining according to the strict (Kruger classification) criteria by two different examiners (intralaboratory blind assessment). Mean percentages of morphologically normal spermatozoa were identical on wet and stained preparation slides (4.79 vs. 4.61, p >.05). Wide divergence of results was found with respect to the percentage of sperm with head and midpiece defects with the two different preparations (p >.001). The percentage of sperm tail defects was similar in both methods (p >.05). Simple linear regression analysis between the two methods revealed very good correlation for the morphologically normal spermatozoa (r =.83), but poor correlation for the sperm head, midpiece, and tail defects (r =.25,.25, and.28, respectively). Wet preparation is suitable only for the morphologically normal spermatozoa, but to determine the percentage of the defective spermatozoa, staining the smear is recommended.
In order to investigate possible DNA damaging effects of ultrasound, the micronucleus assay on cytokinesis blocked human lymphocytes was performed. Preparations were stained by conventional Giemsa staining technique combined with additional staining techniques using fluorescent dye DAPI and silver nitrate. Blood samples were taken from medical personnel employed on ultrasonic scanning in medical diagnosis and unexposed control subjects from general population. Lymphocytes were cultivated in vitro at 37 degrees C. Cytochalasin-B in final concentration of 6 microg/ml was added 44 h after mitogen stimulation and cultures were harvested 28 h thereafter. Staining with both additional techniques can be used to distinguish micronuclei originating from breakage or mitotic loss of certain human chromosomes bearing DAPI-positively stained or silver-positively stained regions. The results obtained indicate statistically significant increases in total number of micronuclei and changes in their distribution in exposed subjects compared to control. Based on different intensity of DAPI staining signal-positive and signal-negative "type" of micronuclei are distinguished, while silver staining has revealed Ag-NOR+ and Ag-NOR- micronuclei. In exposed subjects a prevalence in number of Ag-NOR+ micronuclei over Ag-NOR-micronuclei compared to control was observed, indicating greater susceptibility of chromosomes from D and G groups to damage caused by continuous occupationally exposure to ultrasound. In spite of their limitations, our results indicate that combination of conventional Giemsa staining of micronuclei with fluorescent dye DAPI and silver nitrate staining techniques can be valuable complement to the standard micronucleus assay.
Third and fourth instars of Wyeomyia smithii were reared in Giemsa stain at 4 concentrations between 4 x 10(-7) and 10(-5) g/liter. The mosquitoes retained the blue mark as adults and remained marked throughout their laboratory life. Concentration of Giemsa significantly affected eclosion success but had no significant effect on mean days to pupation or days as a pupa, male or female adult longevity, per-capita female fecundity or fertility. Larval exposure to low concentrations (4 x 10(-7) or 10(-6) g/liter) of Giemsa stain provides an effective lifetime tag for otherwise indistinguishable laboratory populations.