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Kallichrome: a new stain for erythroblasts.

Using the obscure dye kallichrome, erythroblasts at all stages of maturation demonstrated intense yellow to yellow-brown staining of both nucleus and cytoplasm. Staining of this type was not observed in other types of normal or pathological marrow cells. As such, kallichrome may be a valuable stain for identification of erythroblasts and their distinction from other types of blood cells.

Anemia, Pernicious↗

Histochemical techniques to localize rubber in guayule (Parthenium argentatum Gray).

There are few histochemical techniques for staining rubber in the cells of guayule. With regard to specificity, clarity and retention of the stain we found staining with oil red O or iodine-potassium iodide for bright field, and a combination of oil red O and dansyl chloride for epifluorescence microscopy to be the best methods. The plausible mechanisms of staining are discussed.

Azo Compounds↗

The value of fluorescence microscopy of auramine stained sputum smears for the diagnosis of pulmonary tuberculosis.

Laboratory diagnosis of pulmonary tuberculosis rests on the bacteriological examination of sputum smears stained by the Ziehl-Neelsen (ZN) method for acid fast bacilli (AFB). In the present study, we have compared light microscopy of ZN stained smears with that of fluorescence microscopy of sputum smears stained by auramine-phenol flurochrome dye for detection of AFB in sputum specimens. Sputum specimens from a total of 2,600 clinically suspected and diagnosed cases of pulmonary tuberculosis were examined by both the methods. Sputum specimens from a total of 1,104 patients were found to be positive for AFB. These included sputa from 975 (37.5%) patients positive for AFB by both ZN and auramine staining methods and sputa from an additional 129 (4.96%) patients positive for AFB by auramine staining only. Thus auramine staining of sputum smears in comparison to that of ZN staining is a better method of sputum microscopy for demonstration of AFB in sputum specimens. Fluorescence microscopy is relatively more sensitive and has the added advantage of allowing a large number of sputum specimens to be examined in a given time, in laboratories equipped with a fluorescent microscope.

Bacteriological Techniques↗

Phagocytosis and killing of Mycobacterium avium complex by human neutrophils.

Organisms belonging to the Mycobacterium avium complex (MAC) cause life-threatening bacteremia in immunocompromised patients. Monocytes and macrophages are thought to be responsible for ingestion and killing of MAC. However, it has been suggested that neutrophils may play a role in the early immune response to MAC infection. Here, neutrophils in autologous plasma were incubated (at 0 and 37 degrees C) with M. avium labeled with Auramine O, a potent fluorochrome. Neutrophil phagocytosis was measured by flow cytometry. Neutrophils incubated at 37 degrees C showed an increase in fluorescence over time with a maximum at 15 min, whereas neutrophils on ice showed no time-dependent increase in FL1. At 15 min Fl 1 at 37 degrees C was twice as high as FL1 at 0 degrees C. Examination under the fluorescent microscope showed multiple intracellular fluorescent mycobacteria. Results in nine independent experiments showed time-dependent decrease of colony-forming units in neutrophil-associated live M. avium. Significant killing was observed within 30 min and was complete by 120 min. Observation by electron microscopy clearly confirmed the presence of intraphagosomal MAC, both intact and with evidence of degradation. These data demonstrate that MAC is rapidly phagocytized and killed by human neutrophils. The newly established flow cytometry method should be useful in further studies of neutrophil function and of the role of G-CSF and other cytokines in MAC disease.

Adult↗

[Reaction of the seedlings of flowering plants to the effects of imino-di-(paradimethylaminophenyl)-methane-aurine-00].

The influence of auramine-00 on seedlings of three species of plants - Cucurbita pepo L., Helianthus annuus L., Zea mays L.-was investigated. Auramine was added in agar-agar, on which the seedlings were growing. Two concentrations of auramine were used--0.001%. Besides the delay in growth, also the formation of local overgrowth of cotyledons and hypocotyl of C. pepo, the base of cotyledons of H. annuus and mezocotyl of Z. mays were observed. The histological analysis has shown these swellings to arise on the grounds of two processes: pathological proliferation and hypertrophy.

Aniline Compounds↗

[Comparison of auramine-rhodamine and Erlich-Ziehl-Neelsen staining methods for the diagnosis of tuberculosis].

The aim of this study was to compare auramin-rhodamine (A-R) and Erlich-Ziehl-Neelsen (EZN) staining techniques for the diagnosis of tuberculosis. Of 311 sputum samples collected from active pulmonary tuberculosis patients and tuberculosis-suspected patients, 103 (33%) were found culture positive. In the direct microscopic examination of EZN stained smears, 86.4% (89/103) of culture positive samples, and 3.8% (8/208) of culture negative samples yielded asido-resistant bacteria, while these rates were 74.8% (77/103) and 11.5% (24/208) for A-R staining method. When culture was accepted as reference method, the specificity and sensitivity of the staining techniques were found as 88.5% and 74.8% for A-R, and 96.2% and 86.4% for EZN, respectively. As a result, it was concluded that, the use of A-R staining alone, could not be an alternative method to EZN staining.

Benzophenoneidum↗

Comparative study of some factors affecting enumeration of moulds using dilution plate techniques.

The influence of dilution plating technique, nature of diluent, culture media and incubation period on the enumeration of moulds have been studied. Three new culture media containing Auramine, Gentian Violet and Malachite Green respectively have been induced in this study. No significant differences were observed between results obtained after 3, 5 and 7 days of incubation. Significantly higher recoveries were obtained using the surface-spread method than pour plate method. Using the first technique no effect of diluent was observed, and among the different culture media studied higher counts were obtained with medium containing Auramine.

Benzophenoneidum↗

Enhanced detection of Mycobacteria stained with rhodamine auramine at 37 degrees C.

Sputum smear microscopy is the most efficient and rapid technique for detection of acid-fast bacilli (AFB). Fluorochrome method of staining is preferred for Mycobacteria in the overburdened laboratories as the fluorescing bacilli are more readily detected than the fuchsin stained bacilli in shorter period of time. A total of 300 sputum samples obtained from suspected cases of Tuberculosis were collected and were subjected to staining by rhodamine auramine at 37 degrees C and also at room temperature (conventional method). The smears were then blindly evaluated. Fifty-eight samples were positive by both methods and 5 were positive at 37 degrees C only. Staining at 37 degrees C increased the smear positivity by 8.6% over conventional staining at room temperature. No smears were positive only with staining at room temperature alone. Out of 58 smears positive by both methods, 25 had equal number of AFB in both smears, 22 had more AFB in smear stained at 37 degrees C and 11 had greater number of AFB in smears stained at room temperature. Our study, therefore, indicates that rhodamine auramine staining at 37 degrees C is superior to conventional auramine method at room temperature for detecting AFB in sputum smears.

Benzophenoneidum↗

Reduced detection by Ziehl-Neelsen method of acid-fast bacilli in sputum samples preserved in cetylpyridinium chloride solution.

SETTING: Twelve health facilities implementing the DOTS strategy, and the Tuberculosis Research Centre (TRC), Chennai, India. OBJECTIVE: To determine the detection rates using Ziehl-Neelsen (ZN) and auramine-phenol to stain acid-fast bacilli (AFB) in sputum samples stored in cetylpyridinium chloride (CPC) solution. METHODS: Two smears were prepared from each of 988 sputum samples collected in CPC and randomly allocated, one to ZN and the other to auramine-phenol staining. All samples were processed for culture of Mycobacterium tuberculosis. RESULTS: A significantly higher proportion of samples were negative using the ZN method compared to the auramine-phenol method (74.5% vs. 61.8%, McNamara's paired chi2 test; P < 0.001). Among 377 samples that were positive using auramine-phenol, 44% were negative using ZN. There were more culture-positive, smear-negative samples in ZN (52.7%) than in auramine-phenol (30%); the difference attained statistical significance (McNemar's paired chi2 test; P < 0.00004). Using ZN, of the 104 smears made immediately after collection, 52 were positive for AFB, of which only 35 (67.3%) were positive after storage in CPC; the reduction in the number of positive smears attained statistical significance (McNemar's paired chi2 test; P = 0.004). CONCLUSION: Detection of AFB in sputum samples preserved in CPC is significantly reduced using ZN staining.

Benzophenoneidum↗

Increased detection by restaining of acid-fast bacilli in sputum samples transported in cetylpyridinium chloride solution.

SETTING: Seventeen health facilities in a Tuberculosis Unit, and the Tuberculosis Research Centre (TRC), Chennai, India. AIM: Evaluation of restaining by the auramine-phenol method for detection of acid-fast bacilli (AFB) in direct smears of samples transported in cetylpyridinium chloride (CPC) solution. METHODS: Among patients attending the above health facilities, 730 samples were collected in CPC and transported to the TRC. Two direct smears were prepared from each sample, one stained by the usual auraminephenol method (primary staining) and the other stained again by the same method (restaining) for examination by fluorescence microscopy. All the samples were processed for culture of Mycobacterium tuberculosis. RESULTS: A significantly higher proportion (49.6%) of samples were positive by restaining compared to primary staining (32.5%, P < 0.0001). Of 362 samples positive by restaining, 38.7% were negative by primary staining. The yield of different grades of smears was significantly higher with restaining than with primary staining (P < 0.0001). More smear-negative culture-positive smears were observed with the primary than with the restaining method (178/400 [44.5%] vs. 78/ 400 [19.5%], P < 0.001). CONCLUSION: The rate of detection of AFB in direct smears made from sputum samples transported in CPC was higher on restaining than on primary staining.

Bacteriological Techniques↗