Spreading of pseudopodia and cytoplasmic organization in drug-treated macrophages: a study by negative staining technique.
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Animals↗
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Mesosomes observed in unfixed cells of Staphylococcus aureus by negative staining.
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Cell Membrane↗
[Capsular-like structure in B. melaninogenicus as revealed by Gins' negative staining method (author's transl)].
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Bacteriological Techniques↗
The adsorption staining technique applied to isolated premessenger ribonucleoprotein particles: a comparison with conventional techniques using electron microscope tomography.
A specific type of premessenger RNP particle, Balbiani ring granules from the dipteran Chironomus tentans, was biochemically isolated and visualized in three dimensions with electron microscope tomography. The particles were prepared for electron microscopy in three different ways: positively stained, negatively stained and adsorption-stained (embedded in polyvinyl alcohol, PVA, and concomitantly stained). The results were compared with those obtained for RNP particles studied in situ in ultrathin sections of plastic-embedded cells. The positively stained particles were compacted and heavily deformed with little or no internal structure. The negatively stained and the adsorption-stained particles were well preserved; the outer contours and the central cavities of the particles were outlined. The internal structure, i.e. the folded 7-nm elementary fibre, could not be recognized in the negatively stained particles. In the adsorption-stained particles, however, the fibre was discernable, although not quite as distinctly demarcated as in the plastic-embedded samples. We conclude that embedding in PVA with concomitant staining with uranyl acetate is a rapid method to obtain both good preservation and staining of isolated RNP particles. The PVA-embedded particles were also found to be sufficiently resistant to irradiation to permit a comprehensive tilt-series to be taken for electron microscope tomography.
Adsorption↗
Ultrastructure of sarcoplasmic reticulum preparations.
Fragmented sarcoplasmic reticulum (FSR) from rabbit muscle was examined by positive staining, negative staining, and freeze-etch electron microscopic techniques in the absence and presence of calcium transport conditions. The existence of 30-40 A particles covering the outer surface of FSR vesicles was confirmed by two different negative stains in unfixed, glutaraldehyde-fixed and osmium tetroxide-fixed material. Freeze-etch microscopy revealed a second type of particle, 80-90 A in diameter, on the fractured surfaces of FSR vesicles. Following calcium oxalate accumulation, negative and positive staining techniques provided evidence for large nodular deposits within FSR vesicles which probably correspond to calcium oxalate crystals and are responsible for increments in turbidity during calcium oxalate accumulation. The most probable configuration of FSR vesicles in solution is spherical. "Tadpole" or tubular configurations were not seen by freeze-etch microscopy, positive staining, or in prefixed negatively stained material.
Animals↗
Comparison of electron microscopic techniques for enumeration of endogenous retrovirus in mouse and Chinese hamster cell lines used for production of biologics.
Murine myeloma and Chinese hamster ovary cells are used widely in the manufacture of recombinant proteins for biopharmaceuticals. However, rodent cell lines express endogenous retrovirus, which necessitates appropriate design of purification processes to remove virus in excess of the calculated maximum retroviral load. Currently, electron microscopy is the method of choice for determination of retroviral titre in bulk harvest. In this study we compared three electron microscopy techniques to determine retroviral titre in bulk harvest. These were direct negative stain, negative stain after sucrose-density purification and thin section electron microscopy of pelleted supernatant. The study demonstrated that the level of C-type retrovirus associated with cells was predictive of the viral load in cell culture supernatants. The most accurate method for quantifying viral load was direct counting, followed by thin section of pelleted supernatant and negative stain after sucrose concentration. The most practical method was thin section of resuspended pelleted supernatant, which gave improved detection limits.
Animals↗
Ultrastructure, serogrouping and localization of surface antigens of Bacteroides intermedius.
The surface ultrastructure of 21 strains of Bacteroides intermedius was investigated by electron microscopy. Rat monoclonal antibodies (mAbs) were used to define serogroups and to detect the location of surface antigens. All 21 isolates had capsules as demonstrated by the use of wet and dry Indian ink stains. Negative staining of whole cells with 1% (w/v) methylamine tungstate showed that all 21 isolates carried clumped peritrichous fibrils with strain dependent morphology, density and length (less than or equal to 0.75 micron). Fibrils on 11 of 13 fresh clinical isolates were more conspicuously clumped and easily visible, whereas those on 6 of 8 laboratory strains were indistinct and were at the limits of the resolution of the negative staining technique. Staining with ruthenium red (RR), followed by thin sectioning, revealed a dense, amorphous RR staining layer (RRL), up to 24.8 +/- 3.0 nm thick, adjacent to the outer membrane on all of 15 strains examined. All isolates had a less dense RR staining matrix (RRM) extending away from the RRL. The structure of the RRM varied between strains. Four rat mAbs (37BI6.1, 38BI1, 39BI1.1 and 40BI3.2) were used to serogroup the 21 strains of B. intermedius. Immunonegative staining revealed that the mAbs were not directed against fibrilis. Antigens recognized by mAb 37BI6.1 and mAb 39BI1.1 were located on the surfaces of cells, beneath fibrils, and on extracellular vesicles. mAb 38BI1 recognized an antigen which was most accessible on lysed cells, and non-specific binding of mAb 40BI3.2 to grids prevented its localization on the cell surface.
Antigens, Bacterial↗
A "microtubule" in a bacterium.
A study of the anchorage of the flagella in swarmers of Proteus mirabilis led to the incidental observation of microtubules. These microtubules were found in thin sections and in whole mount preparations of cells from which most of the content had been released by osmotic shock before staining negatively with potassium phosphotungstate (PTA). The microtubules are in negatively stained preparations about 200 A wide, i.e. somewhat thicker than the flagella (approximately 130 A). They are thus somewhat thinner than most microtubules recorded for other cells. They are referred to as microtubules because of their smooth cylindrical wall, or cortex, surrounding a hollow core which is readily filled with PTA when stained negatively. Since this is probably the first time that such a structure is described inside a bacterium, we do not know for certain whether it represents a normal cell constituent or an abnormality, for instance of the type of "polysheaths" (16).
Cross-Linking Reagents↗
hSNF5/INI1-deficient tumours and rhabdoid tumours are convergent but not fully overlapping entities.
Rhabdoid tumours (RTs) are rare but highly aggressive tumours of childhood. Their rarity and their miscellaneous locations make the diagnosis particularly challenging for pathologists. Central nervous system and peripheral RTs have been associated with biallelic inactivation of the hSNF5/INI1/SMARCB1 (hSNF5/INI1) tumour suppressor gene. Immunohistochemistry (IHC) with a monoclonal anti-hSNF5/INI1 antibody has recently been proposed as an efficient diagnostic tool for RTs. We have conducted a retrospective study of 55 tumours referred to our institution with a suspicion of RT. This analysis included pathological review, IHC with anti-hSNF5/INI1 antibody, and molecular investigation using quantitative DNA fluorescent analysis and sequencing of the nine exons of hSNF5/INI1. The molecular lesion could be detected in 37 of the 39 cases exhibiting negative staining for hSNF5/INI1. In the two discrepant cases, the lack of detection of genetic abnormality was probably owing to the presence of a high number of non-tumour cells in the samples. This indicates that hSNF5/INI1 IHC is very sensitive and highly specific for the detection of hSNF5/INI1 loss-of-function. Among the 38 cases with typical RT histological features, six failed to exhibit hSNF5/INI1 mutation and stained positive for hSNF5/INI1. This strongly supports the evidence of a second genetic locus, distinct from hSNF5/INI1, associated with RT. Conversely, seven tumours with histological features poorly compatible with RT stained negative for hSNF5/INI1; they nevertheless exhibited an age of onset and a clinical behaviour similar to RT. This suggests that hSNF5/INI1 inactivation is not strictly limited to typical RT but characterizes a wider family of hSNF5/INI1-deficient tumours. Consequently, we believe that anti-hSNF5/INI1 IHC should be performed widely, even when the pathological characteristics are not typical. The molecular investigation should be performed in infants when a rhabdoid predisposition syndrome is suspected.
Adult↗
Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy.
Internalization of CD4 molecules on human CD4-enriched T-cells was demonstrated by immunocytochemical electron microscopy. CD4+ T-cell subclones were obtained from normal human peripheral blood, followed by one-way MLC screening and co-culturing with IL-2. Fixed and non-fixed T-cell samples were indirectly immunolabeled with mouse anti-human CD4 monoclonal antibody and goat anti-mouse IgG conjugated with peroxidase. Unfixed T-cells were immunolabeled at 4 degrees C and then re-incubated for 5-45 min at 37 degrees C. The selected CD4+ T-cell subclones showed strong CD4 binding on the cell surface after IL-2 incubation. However, fresh T-cells, monocytes, bone marrow cells and CD8+ T-cells all stained negative for CD4. The distribution of CD4 molecules on the fixed cell surface showed a homogeneous pattern. Capping and internalization of CD4-antibody-peroxidase complexes from the cell surfaces were observed follow a pathway of receptor-mediated endocytosis in unfixed T cells. Endocytotic vesicles, vacuoles of diverse sizes and shapes near the cell membrane or deep in the cell center were found to contain CD4 molecules. Negatively stained Golgi saccules were observed up to 45 min after re-incubation. These results suggest that increased CD4 molecules can be induced on the surface of normal human T-cells in vitro. Internalization and accumulation of CD4 molecules occurred in CD4-enriched T-cells with IL-2 pretreatment.
Adult↗
Histopathological prediction of hepatitis B recurrence in liver allografts.
The aim of this study was to review the clinical, serological and histopathological features of 20 patients who underwent orthotopic liver transplantation for hepatitis B virus (HBV) related liver disease in an attempt to identify factors which predict recurrence of HBV infection, pattern of disease and disease severity. Hepatitis B recurrence occurred in 11 patients (55%). Positive immunoperoxidase staining for HBsAg and HBcAg of the hepatectomy specimen were shown to have a sensitivity (82% and 64% respectively) and specificity (55% and 66%) in predicting recurrence. This was comparable to serological markers such as HBV DNA (36% sensitivity and 89% specificity). The staining for HBcAg in some cases was weak, focal and had a reticular pattern. Therefore, examination of multiple blocks with a high magnification was necessary to detect HBcAg staining. Negative staining of multiple sections for HBsAg and HBcAg were shown to have a good correlation with clearing of HBV infection (5 out of 6 patients). If recurrence occurred, the range of histological and clinical patterns of HBV recurrence was varied, with severe disease occurring in 5 patients. Neither pre-transplant serology nor hepatectomy immunoperoxidase staining characteristics were shown to be of value in predicting the pattern or the severity of disease once HBV recurrence had occurred. This study indicates that lack of staining for HBV antigens on multiple sections of the native liver may predict a subgroup of HBsAg positive transplant patients who will not suffer from recurrent disease. Once disease did occur however, markers for severity of recurrence were not identified.
Base Sequence↗
Staining nucleic acids and proteins in electrophoresis gels.
This review addresses the most widely used stains for gel electrophoresis in molecular biology today. A brief introduction to electrophoresis is given, followed by a description of agarose and acrylamide gel formation. The major stains for nucleic acids are described, most of which are fluorescent and intercalate into the double helix structure of the nucleic acids. An ultraviolet transilluminator is necessary to visualize the fluorescent bands. The major protein stains include Coomassie blue, silver, and commercial fluorescent stains. Negative staining also can be used to stain the gel background, leaving the protein bands of interest transparent and visible against a white background.
Acrylic Resins↗
Expression pattern of a newly recognized protein, LECT2, in hepatocellular carcinoma and its premalignant lesion.
Leukocyte cell-derived chemotoxin 2 (LECT2) is a recently isolated protein and has been shown to be synthesized by human hepatocytes. All hepatocytes show diffuse immunostaining for LECT2 within the cytoplasm. In the present study, an attempt was made to clarify the expression pattern of LECT2 in nine cases of low-grade malignant hepatocellular carcinoma (LGM-HCC) and five cases of advanced HCC and 19 cases of premalignant lesion, termed atypical hyperplasia (AH), using the indirect immunoperoxidase technique. Variable spotty to coarsely diffuse staining in the majority of cells, a mixture of positively staining and negatively staining areas, and essentially negative staining was observed within the cellular cytoplasm of AH, LGM-HCC and advanced HCC, respectively. The expression of LECT2 became weaker with the progression of multistep hepatocarcinogenesis. The data clearly demonstrate that LECT2 becomes essentially negative in full-blown HCC cells and that the histological distinction between AH and LGM-HCC is valid. It also seems likely that LECT2 is related to hepatocyte growth.
Adult↗
Morphological description of surface structures on strain B41 of bovine enterotoxigenic Escherichia coli bearing both K99 and F41 antigens.
In order to describe morphologically the structures on the cell surface of bovine enterotoxigenic Escherichia coli, variants of reference strain B41 (K99+F41+) either negative for K99 and positive for F41 antigens (variants B41A, B41*C), or phenotypically negative for both antigens (variants B41B1, B41B2, B41*CB), and a transconjugant harbouring the K99 plasmid and expressing the K99 adhesin [transconjugant B41 x H510a:H510(2)] were examined by transmission electron microscopy using negative staining. Several negative staining procedures were tested for strain B41 and variant B41A: direct harvesting of strains into ammonium molybdate (2%, w/v), with bacitracin (50 micrograms ml-1) as wetting agent, gave the best results. Three morphologically distinct structures on the cell surface could be identified in cultures grown on Minca medium. Firstly, thin, filamentous, flexible fibrillar structures, presenting a helical structure and a mean diameter of approximately 3 nm, were recognized as K99 fimbriae, since they were present on strain B41 and on transconjugant H510(2), but not on K99-negative variants nor on the recipient strain H510a. Secondly, coil-like structures with a diameter of about 17-20 nm were observed on strain B41 and on variants B41A and B41*C. These structures appeared to consist of two or more curled filaments (diameter 3 nm) joined to coil on themselves into dense spirals. They were very rare in variants B41B1 and B41B2 and were absent on variant B41*CB and on a transconjugant B41* x B41*CB, which had re-acquired the K99 plasmid and which again exhibited K99 fimbriae. Strains B41 and variant B41A gown at 37 degrees C for 24 h on sheep-blood agar exhibited coiled structures like those seen on Minca medium. In contrast, after growth at 18 degrees C for 48 h (which inhibits the synthesis of F41 antigen), coiled structures were no longer expressed on the cell surface of strain B41 and variants B41A and B41*C. Thus the presence of coiled structures correlated with the expression of F41 antigen in strains and variants, which suggests that F41 had a coiled morphology. Finally, straight fimbriae (diameter 6.5-7 nm) were observed on the cell surface of every strain and variant. Their expression on the cell surface was enhanced by several subcultures in th e static broth, and it was inhibited by subculture on agar, but not by culture at 18 degrees C after serial subcultures in static broth. These facts indicated that the straight fimbriae could be common fimbriae, and excluded their being F41 structures.
Animals↗
Cell adhesion molecule L1 in folded (horseshoe) and extended conformations.
We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.
Cell Adhesion↗
Negative staining-carbon film technique: new cellular and molecular applications.
Two techniques are presented which extend the original negative staining-carbon film technique into new areas of cellular and molecular application. These relate (1) to the production of negatively stained specimens of single-layer plasma membrane split from intact cells during the overall procedure that are negatively stained from the cytoplasmic face and (2) to the production of negatively stained specimens directly from glycerol-containing protein solutions, membrane or viral suspensions. In both cases in vacuo drying onto mica from glycerol is performed, prior to deposition of a carbon film. (For the cellular technique, freshly cleaved mica is firstly rendered positively charged by immersion in Alcian blue.) This is followed by release of the carbon film plus adsorbed membrane or protein by floating onto water, with subsequent negative staining. Selected preliminary applications using human erythrocyte membrane and the high molecular weight (native) human erythrocyte tripeptidyl peptidase-II complex are given and considered speculation as to the future application of the techniques is provided.
Carbon↗
[Electrical properties of the foot-and-mouth disease virus].
It has been shown that the electron microscopy method can be used for characteristics of the electric properties of foot-and-mouth disease virus. The appearance of simultaneous positive and negative staining during the negative staining of virus preparations with 3-4% PTV solution shows the presence of full virions of constant poles designated as positively and negatively stained areas of protein coat surface. The lateral orientation of virions on the film at routine conditions of preparation and the possibility of virion orientation on the film in the external electrical field allow to characterize the full virions as electric dipoles. It has been suggested that there is a relationship between a virus structure and the character of its electric properties.
Aphthovirus↗
Spraying of phenol red dye as a screening test for Helicobacter pylori infection in surgically resected stomach specimens.
BACKGROUND: Endoscopic spraying of phenol red dye and urea (phenol red test) has recently been used to assess the distribution of Helicobacter pylori in the gastric mucosa. We examined whether the phenol red test could be used to detect H. pylori in surgically resected stomachs. METHODS: A total of 82 surgically resected stomachs, obtained from 82 patients (mean age, 60.1 years; range, 33-84 years) with early gastric carcinomas were examined. Phenol red solution and urea were sprayed over the entire mucosa of each resected stomach. A color change from yellow to red was considered as a positive reaction for H. pylori. Gastric mucosal specimens taken from positively stained and negatively stained areas on the phenol red test were then examined immunohistochemically to determine the degree of H. pylori colonization. RESULTS: Diffusely positive reactions were seen in 16 resected stomachs (19%), and regionally positive reactions were seen in 36 (44%). The other 30 stomachs (37%) showed no color change (negative reaction). H. pylori was detected immunohistochemically significantly more frequently in positively stained than in negatively stained areas ( P << 0.0001). Specificity, sensitivity, and predictive values for positive and negative results of the phenol red test, determined on the basis of H. pylori immunostaining, were 100%, 74.3%, 100%, and 72.7%, respectively. CONCLUSIONS: The phenol red test is a specific, relatively sensitive, rapid, easy-to-use, and repeatable method that can be used to diagnose H. pylori infection in surgically resected material. It enables pathologists as well as gastroenterologists with no microbiological expertise to easily diagnose H. pylori infection.
Female↗