Sputum screening by Nomarski interference contrast microscopy.

Gram-stained smears of specimens submitted for sputum cultures were compared with direct wet mounts examined by Nomarski interference contrast microscopy (NIM) for enumeration of squamous epithelial cells (EPC) and leukocytes (WBC). The results obtained by the two methods were comparable, but specimens were more rapidly screened and cell types were more readily differentiated by NIM. Specimens submitted for sputum culture over a 3-month period were examined for EPC and WBC by NIM. Twenty-two percent of the specimens had greater than 25 EPC/field or a predominance of EPC (class I), 30% had greater than 25 EPC and greater than 25 WBC/field (class II), and 48% had greater than 25 WBC/field or a predominance of WBC (class III). The clinical relevance of the culture results was determined by reviewing the records of patients whose specimens were included in the study. Class I specimens provided only 30% clinically relevant culture results. Specimens in class II provided useful culture results in 63% of the patients, and 96% of those in class III provided clinically relevant information. The results confirm the value of sputum screening and demonstrate that NIM provides a rapid, simple, and accurate method for sputum screening.     

Electron microscopy of endothelial cells in culture: III. Freeze-fracturing

Summary Endothelial cells can be examined by freeze-fracturing and replication techniques performed on pellets of endothelial cells scraped off their culture substratum or, alternatively, cells grown on pieces of cover glass can be fractured as monolayers. Deep etching can be performed on either preparation provided that the cryoprotectant glycerol is omitted and water, which sublimes away during the etching process, is substituted.The cleaning and handling of the replicas for subsequent viewing in the electron microscope must be performed with care and are described in detail.     

Acid-fast microscopy on polycarbonate membrane filter sputum sediments.

Polycarbonate membrane filters were used to concentrate 916 sputum specimens for detecting acid-fast bacilli by microscopic examination. These results were compared with those of smears prepared from centrifugates and direct smears of the same specimens. Culture isolation, the control procedure, demonstrated the presence of acid-fast bacilli in 76 specimens. The number of positive specimens detected by microscopy was 82 on polycarbonate membrane filter concentrates, with an 80.2% sensitivity; 53 on centrifugate smears, with a 62.2% sensitivity; and 44 on direct smears, with a 55.8% sensitivity. Acid-fast microscopy results demonstrated that the sensitivity of the polycarbonate membrane filter sputum concentration method was superior to that of the recommended centrifuge concentration method and that the former method may be considered a rapid alternative when culture for acid-fast bacilli is impractical.     

Fc-mediated immune precipitation. III. Visualization by electron microscopy.

Fc-mediated interactions between immune complexes are of major importance for the precipitin reaction. In the present study these interactions were investigated by means of electron microscopy. Keyhole limpet haemocyanin (KLH) was adsorbed to a thin glow charged carbon supporting film and reacted with either rabbit anti-KLH IgG or anti-KLH F(ab')2 fragments. The Fc-Fc interactions were investigated by reacting these surface-adsorbed antibody-rich KLH immune complexes with soluble, antigen-rich ferritin-anti-ferritin complexes using either rabbit anti-ferritin IgG or the corresponding isomolar F(ab')2 fragments as antibody. Fc-Fc interactions were indicated by the formation of clusters or ring structures of ferritin molecules, which were only seen when using KLH anti-KLH IgG and ferritin-anti-ferritin IgG complexes. When F(ab')2 fragments were used as antibody, no reaction between KLH anti-KLH complexes and ferritin-anti-ferritin complexes could be demonstrated.     

DNA from two Orgyia pseudotsugata baculoviruses: molecular weight determination by means of electron microscopy and restriction endonuclease analysis.

Both aqueous and urea-formamide procedures for spreading nucleic acid were employed for electron microscopic studies on the DNAs from the nucleopolyhedrosis bundle virus (NPBV) and the nucleopolyhedrosis single-rod virus (NPSV) both of which are pathogenic for Orgyia pseudotsugata. The molecular weight estimates via electron microscopy were derived by comparison of the mean length values for the double-stranded relaxed circular DNAs with that of SV40 DNA. The aqueous and urea-formamide spreading methods yielded NPSV DNA molecular weight values of 103 × 10⁶ and 104 × 10⁶ daltons, respectively, and molecular weight values of 87 × 10⁶ and 85 × 10⁶ daltons for NPBV DNA. These molecular weights were compared with molecular weight estimates from restriction endonuclease analysis, sedimentation analysis, and renaturation kinetic analysis. DNA located within the NPBV polyhedra but external to virions was characterized by restriction endonuclease analysis and examined by electron microscopy. It was determined to be composed of fragments of random size and to be viral origin.     

Histochemistry and electron microscopy in the diagnosis of small mucous cells in carcinoma of the stomach

One hundred and two cases of poorly differentiated adenocarcinoma were obtained from 71 surgical and 31 biopsy specimens of the stomach. These tumours produced both intracellular and extracellular mucins, which were demonstrated to be neutral mucins, N-acetyl sialomucins, sulphomucins and O-acetyl sialomucins. The majority of tumours (85.3%) secreted two or more kinds of mucins simultaneously, but only a small proportion (14.7%) produced a single kind of mucin. Ultrastructurally, three types of mucin granules were identified; these differed in shape and density. Tumour cells containing intracellular mucins or mucin granules were designated as mucous cancer cells and further subdivided into small, large and goblet mucous cells; tumour cells having neither mucins nor glandular differentiation were designated as undifferentiated cancer cells. These histochemical distinctions, supported by the electron microscopical observations, may be useful to demonstrate early malignant foci in gastric biopsies.     

Preparation of sputum smears for acid-fast microscopy

A method is presented for the preparation of satisfactory smears for acid-fast microscopy that do not contain viable cells of Mycobacterium tuberculosis.     

Improved laboratory safety by decontamination of unstained sputum smears for acid-fast microscopy

Tubercle bacilli may survive in unstained heat-fixed sputum smears and may be an infection risk to laboratory staff. We compared the effectiveness of 1% and 5% sodium hypochlorite, 5% phenol, 2% glutaraldehyde, and 3.7% formalin in killing Mycobacterium tuberculosis present in smears prepared from 51 sputum samples. The smears were decontaminated by the tube and slide techniques. Phenol at 5%, glutaraldehyde at 2%, and buffered formalin at 3.7% for 1 min (tube technique) or for 10 min (slide technique) were effective in decontaminating sputum smears and preserved cell morphology and quantitative acid-fast microscopy results.     

Label-free intracellular transport measured by spatial light interference microscopy

We show that applying the Laplace operator to a speckle-free quantitative phase image reveals an unprecedented level of detail in cell structure, without the gradient artifacts associated with differential interference contrast microscopy, or photobleaching and phototoxicity limitations common in fluorescence microscopy. This method, referred to as Laplace phase microscopy, is an efficient tool for tracking vesicles and organelles in living cells. The principle is demonstrated by tracking organelles in cardiomyocytes and vesicles in neurites of hippocampal neurons, which to our knowledge are the first label-free diffusion measurements of the organelles in such cells.     

Evaluation of erythrocyte shape and status by laser interference microscopy

The area, thickness, and volume of erythrocytes of different types (discocytes, stomatocytes, and echinocytes) from normal subjects and coronary patients were studied by laser interference microscopy. Increase of pH value leading to the stomatocyte-discocyte-echinocyte transformations resulted in a slight decrease of cell volume. In coronary patients, erythrocyte had larger area and volume and exhibited increased aggregation capacity compared to erythrocytes from controls. The results recommend laser interference microscopy as an adequate method for erythrocyte evaluation in laboratory diagnostic measurements. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 3, pp. 357–360, March, 2008