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Negative staining of rat tail tendon collagen fibrils with uranyl formate appears to reveal more detail in the axial banding pattern than any other positive or negative staining method hitherto employed. In addition, uranyl formate and other uranyl solutions appear to reveal fine, closely spaced, longitudinal filaments which may represent the individual tropocollagen molecules.
A prospective study was done to assess the comparative use of acridine orange and Gram stains for detecting false-positive BACTEC blood culture bottles, thereby eliminating unnecessary subcultures. A total of 1049 NR 6A and 7A bottles that were instrument-positive in the BACTEC 660 nonradiometric system, but were Gram stain-negative, had aerobic and anaerobic subcultures as well as an acridine orange stain performed. Only five of 1049 (0.5%) instrument-positive, Gram stain-negative bottles grew microorganisms on subculture. Of these five microorganisms, acridine orange stain detected two. All five microorganisms were assessed not to be clinically significant based on review of the patients' charts. Our data showed that the Gram stain and the acridine orange stain are comparable for detecting false-positive NR 6A and 7A bottles. We conclude that subculture of BACTEC-positive, Gram stain-negative NR 6A and 7A bottles is not necessary.
The morphology of Escherichia coli 0111:B4 lipopolysaccharide (LPS) in aqueous medium was studied by freeze-fracture and negative staining. Samples processed by freeze-fracture showed LPS aggregates that were mainly spherical or elliptical and of rather homogeneous size. Negative staining, however, showed a more heterogeneous population, although globular structures revealed by both procedures had a similar size. Considering the mechanisms involved in the processing of the samples, we suggest that the genuine shape of LPS aggregates is more likely to be globular, thus being artefactual those forms visualized by negative staining, traditionally associated with the organization of LPS in aqueous suspension.
Structural alterations induced in HeLa cells by herpes simplex virus and the mechanism whereby the virus is formed in the nucleus in crystal arrays were studied by electron microscopy with both the usual and negatively stained sections. Aggregates of granular and filamentous material were observed in the cytoplasm of infected cells with both sections. On the other hand, no remarkable alterations in appearance of the cytoplasmic ground substance were observed with the usual sections of infected cells. However, the cytoplasmic ground substance of infected cells when negatively stained consisted of granular material which was different in appearance from the spongy material constituting the cytoplasmic matrix of uninfected cells. In the nucleus of infected cells, complexes consisting of round bodies, amorphous material, aggregates of uniform granules in rows, and viral crystals were often observed near the nuclear membrane in both types of sections. Examinations of the granular aggregates with negatively stained sections suggested that each granule represents a subunit and that the several adjoining subunits (approximately eight) constitute the requirement for formation of a single viral capsid with a core. Thus, rapid and simultaneous formation of the core and capsid within the aggregate would replace the rows of the granules with the viral crystal. The advantages of negative staining of thin sections for visualization of fine structural alterations are discussed.
Depending on the method of preparation, the actin-rich lamellipodia of motile cells can show very different structural organizations. This situation has been a main contributor to differences in current ideas about the possible mechanisms of cell movement. We have here analyzed the structure of the lamellipodium in whole-mount cytoskeletons using one of the most rapid of crawling cells, the fish keratocyte, employing two procedures considered least damaging to actin filament arrays: freeze-drying and negative staining. At the front of the lamellipodium, where filaments density is the highest, freeze-dried images conveyed the impression of a cross-linked network of very short, interconnected filaments--as previously observed by others--whereas the same regions appeared as a diagonal meshwork of long, more or less straight filaments after negative staining. In general, the linearity of actin filaments was not preserved after freeze-drying, except in situations where the filaments had partially dried down onto the substrate before freezing. In the mid and posterior regions of the lamellipodium the actin filaments appeared to be up to several micrometers long by negative staining, whereas their length was impossible to discern by freeze-drying, owing to filament kinking and aggregation and to the nature of the contrasting procedure, which reveals only the upper layers of filaments. We conclude that while freeze-drying preserves the overall three-dimensional structure of the lamellipodium it also introduces fine-structural distortions in actin that obscure actin filament order. Drying in negative stain appears to stabilise the actin network.(ABSTRACT TRUNCATED AT 250 WORDS)
A negative staining technique is presented based on the use of 40-60 nm quartz membrane supported by a silicon grid. The quartz membrane is fabricated by thermal growth of silicon dioxide on a silicon substrate followed by an anisotropic silicon etching step giving rectangular holes in the silicon substrate. The hydrophilic membrane is shown to be ideally suited for negative staining due to its spreading characteristics, homogeneity, heat resistance and mechanical stability. Micrographs of phage lambda are presented showing the detailed structure of the tail. A simple method of calculating the number of adsorbed particles based on diffusion limited association is also presented.
Sucrose-gradient-purified dictyosomes of plant Golgi apparatus appear, after glutaraldehyde stabilization, as stacks of highly fenestrate and tubate cisternae when negatively stained with phosphotungstic acid, shadowed with heavy metal, or OsO(4)-stained in thin section. The tubular proliferations (diameter 200 to 400 A) extend for several microns from the central region and are united at intervals into an anastomosing network. Associated with the tubules are two kinds of vesicles which are distinguishable on the basis of texture, size, shape, and staining characteristics. One vesicle type is rough-surfaced, nearly spherical, and of uniform dimensions (diameter approximately 600 A). Metal shadowing shows that these vesicles remain spherical after drying. The other vesicle type is smooth-surfaced and varies in both size and shape. Intercisternal elements are revealed, by negative staining, on the surface of internal cisternae after fragmentation of the dictyosome. The progressive differentiation of cisternae from the forming face to the maturing face is observed in thin sections of these isolated preparations. The morphological characteristics observed in negatively stained dictyosomes indicate regions of functional specialization within the dictyosome cisternae and reveal a dictyosome structure more extensive than that envisioned from sections.
Several representative examples are given of the successful application of negative staining across the holes of holey carbon support films using 5% (w/v) ammonium molybdate solution containing trehalose. The inclusion of 0.1% (w/v) trehalose is considered to be most satisfactory, although good data have also been obtained in the presence of 0.01 and 1.0% (w/v) trehalose. The examples given fall into the following groups: protein molecules in the absence of polyethylene glycol (PEG), protein molecules in the presence of PEG (Mr 1000), lipoproteins, lipids and membranes, filaments and tubules, viruses in the absence of PEG, viruses in the presence of PEG, aqueous polymer solutions, and finally for comparison purposes, four unstained samples studied in the presence of trehalose alone. In all these cases, and many others not documented here, successful spreading of the sample across holes has been achieved, with the sample embedded within a thin film of air-dried ammonium molybdate+trehalose. These specimens can be rapidly produced and provide an alternative to negatively stained specimens on carbon support films. Specimen stability in the electron bean is good and such specimens can usually generate superior negatively stained TEM images without flattening and adsorption artefacts. The formation of 2-D arrays/crystals of protein molecules and viruses, suspended across holes in the presence of ammonium molbybdate+trehalose, and trehalose alone, is also demonstrated.
Low-dose electron diffraction of thin single crystals of catalase that are negatively stained with the light-atom compound, dipotassium glucose-1,6-diphosphate, reveals Bragg reflections extending to 4.0A (= 0.40 nm). Under the same conditions, negative staining with the traditional heavy-metal salt, ammonium molybdate, also gives diffraction spots extending to 4.0 A. These results establish that negative staining of protein crystals preserves periodic structural information into the high-resolution range, unlike the widely accepted current belief that this methodology can give a resolution limited to only 20-25 A.
A versatile negative-staining ribonuclease zymogram is described. The method has several advantages as it combines, by means of different staining procedures, high resolving power, sensitivity, and specificity with a rapid, reproducible, and simultaneous analysis of purity of ribonuclease samples on the same polyacrylamide gel. Activity bands can be visualized at any time during the incubation process without staining of the gel. This allows the choice of different staining procedures after incubation. Using poly(C) as substrate less than 1 pg of bovine pancreatic ribonuclease A was detected in less than 2 h after the electrophoretic run. An additional advantage with respect to other methods is that no refrigeration is needed during electrophoresis.
The cross striation of native and reconstituted collagen fibrils is believed to conform to a unique D-band pattern independently of the genetically distinct types of fibril-forming collagens. This investigation focuses on type II native collagen fibrils, whose negative-staining patterns are shown to differ from the usual banding exhibited by type I collagen fibrils. Negative staining with phosphotungstic acid, pH 7.4, was carried out on a) unfixed and b) glutaraldehyde-fixed collagen fibrils isolated from bovine hyaline cartilages. The band patterns obtained and their microdensitograms were compared to similarly processed type I collagen fibrils isolated from bovine fibrous tissues. Only minor differences were observed in unfixed fibrils. In the intraperiod light zones of type II fibrils, two dark bands (interbands X2-Y4 and Y4-Y2) showed different intensities with respect to their homologous bands in type I fibrils. In contrast, a marked difference was shown by glutaraldehyde-fixed fibrils. In comparison with type I fibrils, the greater stain exclusion capacity of type II fibrils yielded both the appearance of supernumerary bands, which altered banding in two intraperiod regions, and differences in the intensity of several bands in three intraperiod regions where the band distribution was similar. This stain exclusion pattern may be accounted for by molecular extradensity. The possibility that it depends on linkage with a higher number of glutaraldehyde residues and/or the persistence of cross-linked collagenic or non-collagenic proteins is discussed. To refer to the glutaraldehyde-induced band patterns in negatively stained type II and type I collagen fibrils, the terms "bands GA(II) 1-12" and "bands GA(I) 1-15," respectively, are proposed.
Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 mum. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per mum(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per mum(2) and 390 +/- 170 per mum(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.
A simple method of obtaining analogue images from traced models of biological specimens is presented. It consists of the photographic defocusing of traced models and it is illustrated with negatively stained cylindrical forms of the ASFV; the black lines of the trace in the model correspond to the negative stain surrounding the viral morphological subunits as seen in the electron micrograph. The photographic defocusing is the means by which the traced model is filtered and is used to introduce grey levels on an otherwise black and white image. The right amount of defocusing is attained when the width of the trace of the model equals the width of the rim of the negative stain appearing between the morphological subunits in the electron micrograph.
The two most common homologous phenotypes (SS and FF) of human placental alkaline phosphatase were purified and observed in the electron microscope by rotary shadowing and negative staining techniques. In the rotary shadowing technique, the molecules of the two phenotypes appeared to be approximately elliptical with slit-like structures in the center of the molecules, suggestive of the groove between two subunits. The dimensions of the rotary-shadowed molecules were calculated as 10.1 nm X 5.7 nm for SS and 10.1 nm X 5.6 nm for FF phenotypes. The negative staining technique delivered more fine detail of the molecules than rotary shadowing. The predominant shape of the molecules in this method appeared to be rectangular, with a longitudinal stain-filled groove and with each of the half molecules (presumably 65,000 Mr subunit) very often appearing bi-lobed. This accounts for the molecules which appear to have four pronounced electron-transparent regions. The dimensions of the negatively stained rectangular-shaped molecules were measured as 7.5 nm X 5.5 nm for SS and 7.6 nm X 5.4 nm for FF phenotypes. No significant difference in electron microscopic appearance between the SS and FF phenotypes were observed.
The ultrastructure of chromatin fibers isolated from erythrocyte nuclei of Necturus maculosus and contrasted with a number of negative stains is described. Long (greater than 1000 nm) fibers are prepared under ionic conditions that promote fiber integrity, fixed with glutaraldehyde and negatively stained with aurothioglucose, ammonium molybdate, methylamine tungstate, sodium phosphotungstate, uranyl acetate and a uranyl acetate-sodium phosphotungstate sequence. All stains yield images of '30 nm' chromatin fibers, but aurothioglucose gives the most consistent diameter measurements (33 nm, S.D. 3.5 nm), and provides the clearest images of individual nucleosomes. Regions of fiber showing structural order are seen with all stains. The most commonly observed is a regular pattern of oblique cross-striations consistent with the visualization of the 'top' or 'bottom' of a helical structure. There is a significant relationship between fiber diameter and the cross-striation angle, consistent with an extensible chromatin fiber. Examination of power spectra prepared from selected ordered regions confirms the visual impressions, and indicates a striation spacing ranging from 11 nm to 18 nm, and dependent on the stain type. Fibers allowed to unfold slightly in a buffer containing 50 mM monovalent ions show evidence of a two-stranded helix-like organization. These results are discussed in terms of current models for the structure of the chromatin fiber.
We have used a combination of centrifugation onto electron microscope grids and negative staining to study the structure of isolated mammalian centrioles. The technique relies on visualisation of structural detail by use of a goldthioglucose negative stain. The approach provides an easy structural definition of the mature and immature centriole and has revealed some novel proximal projections on the mature centriole. The rapid technique should prove of use in future analyses of centriolar structure and biochemistry.
Semi-isolated annulate lamellae were prepared from single newt oocytes (Triturus alpestris) by a modified Callan-Tomlin technique. Such preparations were examined with the electron microscope, and the negative staining appearance of the annulate lamellae is described. The annulate lamellae can be detected either adhering to the nuclear envelope or being detached from it. Sometimes they are observed to be connected with slender tubular-like structures interpreted as parts of the endoplasmic reticulum. The results obtained from negative staining are combined with those from sections. Especially, the structural data on the annulate lamellae and the nuclear envelope of the very same cell were compared. Evidence is presented that in the oocytes studied the two kinds of porous cisternae, namely annulate lamellae and nuclear envelope, are markedly distinguished in that the annulate lamellae exhibit a much higher pore frequency (generally about twice that found for the corresponding nuclear envelope) and have also a relative pore area occupying as much as 32% to 55% of the cisternal surface (compared with 13% to 22% in the nuclear envelopes). The pore diameter and all other ultrastructural details of the pore complexes, however, are equivalent in both kinds of porous cisternae. Like the annuli of the nuclear pore complexes of various animal and plant cells, the annuli of the annulate lamellae pores reveal also an eightfold symmetry of their subunits in negatively stained as well as in sectioned material. Furthermore, the annulate lamellae are shown to be a site of activity of the Mg-Na-K-stimulated ATPase.
A rapid, semiquantitative method for screening large numbers of virus samples by negative staining electron microscopy is presented. Results obtained by this method are compared with results obtained by the pseudoreplica method and a measureddrop method. A figure is presented which represents the limit of detectability for virus by negative staining.