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Results for “Negative Staining”

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PubMed1993

Glutaraldehyde-induced D-band pattern of type II collagen fibrils as revealed by negative staining.

A study was carried out on the negative staining band patterns (1% phosphotungstic acid (PTA) in phosphate buffer, 0.1 M, pH 7.4) exhibited by type II collagen fibrils after treatment with glutaraldehyde (5% in phosphate buffer 0.1 M, pH 7.4) and a comparison was made with the negative staining patterns of glutaraldehyde-fixed type I collagen fibrils. A characteristic D-band pattern was observed for type II collagen fibrils. The gap/overlap ratio was unusually low, with a 0.40 long gap zone and a 0.60 D overlap zone. This banding displayed eleven major light bands instead of the fifteen bands per period observed in the type I collagen patterns. On comparing the two types of microdensitograms, seven negative peaks (light bands) coincided and among them were the peaks corresponding to the N-terminal and C-terminal telopeptide regions. Although less numerous, the negative peaks of type II fibril traces were broader and more marked than those of type I microdensitograms and this feature accounts for the greater stain exclusion capacity of the type II fibrils. This is consistent with the larger quantity of hydroxylysines in type II collagen and, perhaps, with the more abundant hydroxyprolines.

Animals↗

PubMed1987

Electron-irradiation-induced flattening of negatively stained 2D protein crystals.

The thickness of negatively stained 2D crystalline arrays of the bladder membrane does not vary significantly during air drying and exposure to high vacuum. High-dose electron irradiation reduces the thickness to about 60% of the native value. These results, together with the fact that the same behaviour has been observed on another 2D system (gap junctions), indicate that the flattening induced by an electron beam on 2D crystals may be general. The implications for 3D reconstruction of negatively stained objects are discussed.

Animals↗

PubMed2000

Morphology of sodium deoxycholate-solubilized apolipoprotein B-100 using negative stain and vitreous ice electron microscopy.

The primary and secondary structures of apolipoprotein B-100 (apoB-100) are well established. Previous morphological studies have suggested that apoB is a long, flexible, threadlike molecule that encircles the low density lipoprotein (LDL) particle. Several large domain regions of the protein have been observed in frozen hydrated LDL and may be involved in anchoring of the protein to the lipid surface of LDL. Calorimetric studies of sodium deoxycholate (NaDC)-solubilized apoB indicated a similar number of independently melting domains. We therefore undertook a morphological study of NaDC-solubilized apoB-100 using negative stain and vitreous ice cryoelectron microscopy, a nonperturbing preservation technique. Negative staining experiments were performed in two ways: 1) grids were pulled through NaDC-containing buffer surfaces on which monolayers of apoB had been promoted, or 2) apoB molecules were allowed to diffuse onto carbon surfaces of grids that were floated on sample droplets. Vitrified molecules of apoB were obtained by plunging a thin fluid layer of protein adhered to a holey carbon-coated grid into supercooled ethane and by preserving the molecules in liquid nitrogen. The majority of molecules prepared in negative stain and vitreous ice were curved or arced and had alternating thin and thick regions. In negative stain, the apoB molecules lay on the grid perpendicular to the electron beam and had a mean length of 650 A. In vitreous ice the molecules were randomly oriented and their images ranged from 160 to 650 A in length. Vitrified molecules provided visualization of one or two beaded regions. Similar regions were observed in negative stain but the overall thickness was two to three times greater. Some vitrified molecules contained ribbon-like portions. Our study supports previously obtained data on molecule length but suggests that negative staining overestimates molecule width. These first images of vitrified NaDC-solubilized apoB-100 confirm the long, flexible, beaded thread morphology of the molecule and support the unique potential of this technique when coupled with proper molecule orientation and antibody labeling to correlate the tertiary structure of apoB seen in the intact particle with that of the isolated molecule.

Apolipoprotein B-100↗

PubMed1983

Band patterns seen by electron microscopy in ordered arrays of bovine and human fibrinogen and fibrin after negative staining.

When fibers of fibrin clots or fragments of fibrinogen pellets are negatively stained they exhibit in the electron microscope characteristic patterns of cross-striations or bands. Those found in pellet material are indistinguishable from those seen in thrombin-induced fibrin fibers. The pattern seen in fibrin from bovine sources contains three equally spaced faint bands between every two of the broad prominent ones, spaced 23 nm apart. Human material shows a different pattern, one wherein no central faint band is seen, whereas the two remaining ones are broader. Its character is unaffected by crosslinking following fiber formation and preceding negative staining. The bovine pattern, however, is converted by such crosslinking to one that closely resembles the human. It is suggested that the striation pattern in human fibrin is due to juxtapositions of E domains of the parallel-aligned fibrin monomers with tightly coiled COOH-terminal regions of beta and gamma polypeptide chains, with no discernible contribution to the pattern from the alpha chain. In negatively stained untreated fibers of bovine fibrin, however, it is proposed that the COOH-terminal region of the alpha chain becomes tightly coiled, thereby contributing the faint central striation to the band pattern. Crosslinking prevents this conformational change in the alpha chain.

Animals↗

PubMed1984

The pleated sheet: an unusual negative-staining method for transmission electron microscopy of biological macromolecules.

A novel method for preparing negatively stained specimens is described which appears to improve the routine resolution of biological structure in direct images obtained by transmission electron microscopy. In the new method, which we term the pleated sheet technique, macromolecules are adsorbed to a carbon film by the Valentine procedure (R. Valentine, B. Shapiro, and E. Stadtman (1968) Biochemistry, 7, 2143-2152), and the film then carefully pleated while in contact with a 1% uranyl formate solution to trap stain within the folds of pleats. A grid is placed on the compressed film, and film plus grid retrieved with a Saran Wrap drum. Subsequent dehydration produces a filmed grid containing negatively stained macromolecules within the folds of pleated regions and positively stained macromolecules in single sheet regions. The effect of sandwiching sample and stain between carbon layers is to produce exceedingly uniform negative staining so that stain contours more accurately and more reproducibly reflect true molecular contours. Electron micrographs of IgG and IgA molecules prepared by these methods are exhibited that permit unambiguous comparison of structure imaged in the electron microscope against known structures solved by single-crystal X-ray diffraction. Correlation is excellent; the smallest resolvable element in micrographs is an immunoglobulin domain, whose molecular weight is 12 000.

Animals↗

PubMed1977

Electron microscopy of negatively stained and freeze-etched high density lipoprotein-3 from human serum.

High density lipoproteins of d = 1.12 to 1.21 g/ml from human serum (HDL3) were studied by electron microscopy with both negative staining and freeze-etching techniques. For the negatively stained specimens, a modified conventional transmission electron microscope as well as a scanning transmission electron microscope were used. The freeze-etched specimens were examined by a conventional transmission electron microscope. The diameter of HDL3 was found to be 105 +/- 4 A by freeze-etching and 94 +/- 6 A by negative staining. The surface of the HDL3 particles exhibited about 12 discrete domains, 28 +/- 3 A (freeze-etched) and 28 +/- 4 A (negatively stained) in diameter, of undefined chemical composition. Moreover, the freeze-etched specimens revealed an inner core 40 +/- 2 A in diameter, corresponding to estimated values reported previously. All information is consistent with the HDL3 model proposed by B. W. Shen, F. J. Kézdy, and A. M. Scanu [(1977) Proc. Natl. Acad. Sci. USA 74, 837-841], with additional evidence for well-defined surface substructure. The consistency of the images obtained with the various electron microscopy techniques and the marked change in the appearance of the surface in the HDL3 preparations that were digested by phospholipase A2 (EC 3.1.1.4) support the validity of the interpretation.

Freeze Etching↗

PubMed1980

Electron microscopy of negatively stained and unstained fibrinogen.

Electron microscope images of negatively stained fibrinogen are predominantly asymmetric rods 450 A in length and about 60 A in width. The molecules appear to have considerable flexibility, and mass distribution along the major axis is not uniquely distinguished despite apparent beading in some particles. Scanning transmission electron microscopy of unstained fibrinogen again demonstrates that a majority of molecules are rodlike. The results differ from those obtained by negative staining in that a substantial fraction of images are trinodular with striking resemblance to those obtained by C. E. Hall and H. S. Slayter [J. Biophys. Biochem. Cytol. (1959) 5, 11--16] using the mica replica technique. The above results were obtained on glow-discharged carbon substrate films by a simple low-concentration, long-attachment-time modification of standard deposition methods that is diffusion controlled and depends on concentration and time but is independent of pH, buffer, and other staining conditions. Evidence is presented that standard attachment procedures result in artifactual images. Any models of fibrinogen in solution consequently must encompass properties that permit its visualization as an asymmetetric rod by electron microscopy as first suggested by Hall and Slayter 20 years ago.

Animals↗

PubMed1990

Negative staining EM for the detection of Epstein-Barr virus in oral hairy leukoplakia.

The performance of two different EM techniques applied for the detection of Epstein-Barr Virus (EBV) in oral hairy leukoplakia (HL) was assessed, i.e. the conventional two-step method of negative staining (CNS) and negative staining after Airfuge enrichment (ANS). Scrape specimens from the lateral borders of tongue of 66 HIV-positive patients with or without HL, of 3 patients with infectious mononucleosis and of 10 HIV-negative patients were evaluated. While CNS resulted in virus detection only in 25% of clinically diagnosed HL cases, EBV was detected by ANS in 85% of clinically suspected cases of HL. Scrape specimens of individuals negative for HIV were negative in EM while 2 of 3 mononucleosis patients were positive without clinical evidence for HL. Due to this high sensitivity the method of negative staining after Airfuge enrichment appears to be useful in the diagnosis of HL. The finding of EBV in clinically normal oral mucosa in HIV-seropositive individuals is interesting and indicates that EBV expression may precede the clinical appearance of HL.

Adult↗

PubMed1989

Cardiac gap junctions and gap junction-associated vesicles: ultrastructural comparison of in situ negative staining with conventional positive staining.

By comparing in situ negative staining of mammalian heart muscle using La(NO3)3 with conventional positive staining by uranium and lead salts, we showed that 1) the membrane area of rat cardiac gap junctions (GJs) at the intercalated disks is threefold to fourfold greater than previously thought; 2) connexon arrays of cardiac GJ are subdivided into microdomains by connexon-free aisles; 3) profiles of GJ-associated vesicles (GJAVs) of plasmalemmal origin (which are present extracellularly and sharply localized at three extracellular sites) are paired to form GJs with each other and with myocyte plasmalemma; 4) some GJAVs contain arrays of assembled connexons; and 5) myocytes contain intracytoplasmic complexes lying within cylindrical or cigar-shaped membranes and consisting of GJs and multiple vesicles apparently dissociating from these GJs.

Animals↗

PubMed1976

The use of freeze-fracture and negative staining techniques to study the ultrastructure of melanosomes isolated from B16 melanoma.

Freeze-fracture negative staining methods were used to investigate the structure of melanosomes isolated from B16 melanoma. The findings demonstrated the presence of a melanosomal double-layered membrane. Negative staining procedure showed that the melanosomal membrane is sometimes ruptured, displaced or peeled off during the purification process. Fractured melanosomes showed melanin spheres with a mean diameter of 24 nm. This compares with a mean diameter of 26 nm for the melanin spheres revealed by negative staining. The granular appearance of fractured mature melanosomes is due to the fracture following a random path between melanin spheres.

Animals↗

PubMed1991

New version of the negative stain.

We have developed a new version of the negative stain which is very quick, reliable, and easy to perform and which uses a waterproof marking pen instead of nigrosin. It is ideal for teaching the negative staining technique to beginning student microbiologists.

Bacteriological Techniques↗

PubMed1997

Flexibility within myosin heads revealed by negative stain and single-particle analysis.

Electron microscopy of negatively stained myosin has previously revealed three discrete regions within the heads of the molecule. However, despite a probable resolution of approximately 2 nm, it is difficult to discern directly consistent details within these regions. This is due to variability in both head conformation and in staining. In this study, we applied single-particle image processing and classified heads into homogeneous groups. The improved signal-to-noise ratio after averaging these groups reveals substantially improved detail. The image averages were compared to a model simulating negative staining of the atomic structure of subfragment-1 (S1). This shows that the three head regions correspond to the motor domain and the essential and regulatory light chains. The image averages were very similar to particular views of the S1 model. They also revealed considerable flexibility between the motor and regulatory domains, despite the molecules having been prepared in the absence of nucleotide. This flexibility probably results from rotation of the regulatory domain about the motor domain, where the relative movement of the regulatory light chain is up to 12 nm, and is most clearly illustrated in animated sequences (available at http://www.leeds.ac.uk/chb/muscle/myosinhead.htm l). The sharply curved conformation of the atomic model of S1 is seen only rarely in our data, with straighter heads being more typical.

Animals↗

PubMed2000

The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes.

We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections. From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization. In contrast to the two-dimensional map, the tomographic reconstruction of Halob. salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof. volcanii envelopes, although the main features of the lattice and the morphological units are evident. The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation. This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof. volcanii and Halob. salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof. volcanii) and low-dose cryo-tomography (Halob. salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures.

Archaeal Proteins↗

PubMed1997

The conformation of packaged bacteriophage T7 DNA: informative images of negatively stained T7.

Within the icosahedral protein outer shell of bacteriophage T7, a 40-kbp DNA genome occupies a cavity also occupied by a protein cylinder that projects into the DNA from the outer shell. However, neither the internal cylinder nor separately resolved DNA segments are revealed in the conventional negatively stained specimens of intact bacteriophage T7. In the present study, a procedure of negative staining is used that reveals both internal proteins and separately resolved segments of packaged DNA during electron microscopy of intact particles of a hybrid T7 bacteriophage; the hybrid is genetically T7, except for a tail fiber gene that has a segment from the T7-related bacteriophage, T3. The negatively stained packaged DNA segments of this hybrid bacteriophage are found to be wrapped around the axis of the internal cylinder. To obtain additional information about the conformation of packaged T7 DNA, electron microscopy is performed of negatively stained capsids that are incompletely filled with DNA (ipDNA-capsids); a procedure is described for improved isolation of ipDNA-capsids from lysates of hybrid bacteriophage T7-infected cells. The packaged DNA segments of ipDNA-capsids are found not to be wrapped around any axis. Images of ipDNA-capsids are explained by the hypothesis that DNA does not achieve its wrapped condition until the capsid is more than 40% full of DNA. Wrapping via folding is, therefore, proposed to explain the images of DNA packaged in bacteriophage T7.

Bacteriophage T3↗

PubMed1975

Negative staining of rat tail tendon collagen fibrils with uranyl formate.

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.

Acetates↗

PubMed1994

Subcultures of BACTEC-positive but gram or acridine orange stain-negative NR 6A and 7A blood culture bottles are unnecessary.

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.

Acridine Orange↗

PubMed1993

Visualization of lipopolysaccharide aggregates by freeze-fracture and negative staining.

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.

Escherichia coli↗

PubMed1971

Mechanism of intranuclear crystal formation of herpes simplex virus as revealed by the negative staining of thin sections.

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.

Acetates↗