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PubMed1969

A comparison of chloroplast membrane surfaces visualized by freeze-etch and negative staining techniques; and ultrastructural characterization of membrane fractions obtained from digitonin-treated spinach chloroplasts.

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.

Cell Membrane↗
PubMed1985

Light-harvesting chlorophyll a/b-protein: Three-dimensional structure of a reconstituted membrane lattice in negative stain.

The three-dimensional structure of a negatively stained hexagonal membrane lattice containing the light-harvesting chlorophyll a/b-protein complex and phospholipids has been determined to 30-A resolution by image reconstruction from electron micrographs. This lattice has p321 symmetry, a lattice constant of 125 A and a thickness of 75 A. The monomer is shown to be an elongated molecule about 65 A long in the dimension perpendicular to the plane of the membrane. It spans the hydrophobic domain of the membrane in an asymmetric fashion, projecting [unk]20 A from one surface and less from the other. On the basis of this image and available biochemical data, the structure of the complex in the native thylakoid membrane is proposed.

Journal Article↗
PubMed1966

The microcrystalline structure of cellulose in cell walls of cotton, ramie, and jute fibers as revealed by negative staining of sections.

With a new technique of negative staining of sections, it has been possible to observe directly, in ultrathin sections under the electron microscope, the original microcrystalline and microfibrillar structure of cellulose as it occurs in living cells. This method has advantages over the study of isolated fibers used so far by others, in that the original arrangement of microfibrils is better preserved, and their collapse into larger fibrillar units is prevented. With this method, the cell walls of ramie, jute, and cotton fibers have been studied. The size (diameter, 25 to 40 A) and the longitudinal periodicity observed in the single microfibrils and the orientation and spatial arrangement of the microcrystallite within the microfibrils are found to correspond with the latest models derived by others from data obtained by indirect methods such as X-ray diffraction. The microfibril size of about 35 A, found by measuring these structures in sections, agrees with the latest conclusions reached by others in recent work with isolated fibrils.

Cell Wall↗
PubMedDate not supplied

[Rapid detection of viruses by electron microscopy. Interest of negative staining for the diagnosis of some skin lesions of viral origin (author's transl)].

Viral particles can be visualized by electron microscopy using negative staining. Such an approach, widely used in research, provides a method for detecting virions which are present in clinical specimens. It is considered to be the method of choice in cases with suspicion of smallpox. Direct visualization has been systematically applied for the diagnosis of viral diseases in dermatology. Negative staining by pseudoreplication appears to be simple, rapid and efficient. It was thus possible to detect viruses in vesicle fluids, in scrapings and in crusts. During the course of this study 66 cases have been analyzed and viruses have been demonstrated in 49. If a skin lesion seems to be of viral origin, direct examination may confirm the clinical impression.

Humans↗
PubMed1981

Effects of n-alkanes on the morphology of lipid bilayers. A freeze-fracture and negative stain analysis.

The effect of n-alkanes on the ultrastructure of lipid bilayers has been investigated using freeze-fracture and negative stain electron microscopy. It has been found that the morphology of bilayers containing the long alkane tetradecane is quite different from bilayers containing the short alkane hexane. The smooth fracture faces of gel and liquid crystalline state bilayers are unmodified by tetradecane. However, hexane dramatically alters the hydrophobic bilayer interior, producing large (20 to 50 nm) mounds and depressions in the fracture faces. The fracture steps in these multilayer preparations containing hexane are variable in thickness and often considerably wider than the corresponding fracture steps in multilayers which contain tetradecane or are solvent-free. Alkanes also modify the structure of the P beta' or 'banded' phase of phosphatidylcholine bilayers. The incorporation of tetradecane removes the banded structure from both the bilayer's hydrophilic surface, as viewed by negative staining, and the bilayer's hydrophobic interior, as viewed by the freeze-fracture technique. These results are consistent with X-ray diffraction data which imply that long alkanes are primarily located between adjacent lipid hydrocarbon chains in each monolayer of the bilayer, while short alkanes can partition into the geometric center of the bilayer between apposing monolayers.

Alkanes↗
PubMed1978

Negative staining of freeze-fractured envelopes of Escherichia coli K12.

Envelope fragments of E. coli K12 have been produced by freeze-fracturing "by hand" and negatively stained after thawing. The outer leaflet of the plasma membrane disintegrated upon thawing whereas the outer leaflet of the outer membrane did not. Negative staining revealed the following structural features on the outer membrane fragments: (i) "grooves" 4-6 nm wide, (ii) spherical particles 6-8 nm in diameter, (iii) "black dots" 3-8 nm in diameter. Treatment of cells with EDTA before freeze-fracturing caused dilation of grooves into holes eventually leading to fragmentation of the outer membrane. A mutant strain deficient in two outer membrane proteins fractured exclusively through the outer membrane. The outer leaflets so obtained disintegrated upon thawing similarly as observed for the outer leaflet of the plasma membrane.

Cell Membrane↗
PubMed1995

The covering method: an improved negative staining method for ultrathin cryo-sections of tissue.

Ultrathin cryo-sections of biological tissues for electron microscopy provide numerous advantages in cytochemical and morphological studies. However, difficulties still remain in the application of negative staining to cryo-sections. This paper describes a method for negative staining of cryo-sections by covering the grid with a piece of either Collodion or Formvar film. By doing so, appropriate amount of staining solution is retained on the grid for positive effects. Also, the present method requires only a few seconds to stain the ultrathin cryo-sections while previously published methods took up to 10 min.

Animals↗
PubMed1966

Structure of a marine bacteriophage as revealed by the negative-staining technique.

Valentine, Artrice F. (Georgetown University, Washington, D.C.), Peter K. Chen, Rita R. Colwell, and George B. Chapman. Structure of a marine bacteriophage as revealed by the negative-staining technique. J. Bacteriol. 91:819-822. 1966.-The morphology of a marine bacteriophage has been determined by negative-staining techniques and electron microscopy. The virus possesses a head, 600 A in diameter, and a tail which may be from 860 to 1,000 A in lenght. No tail sheath is seen. The appearance of the terminal tail structure is discussed.

Bacteriophages↗
PubMed1996

Negative staining of proteins in polyacrylamide gels with methyl trichloroacetate.

This paper describes a new, sensitive (in the nanogram range), and rapid (two-step) technique for the negative staining of proteins in polyacrylamide gels in the presence or absence of sodium dodecyl sulfate. After separation, gels are incubated with 8% methyl trichloroacetate ester in 38% isopropanol and then washed in water to produce a negative image of colorless proteins against an opaque background. The technique allows unmodified proteins to be recovered for biological studies or transblot for amino acid sequence. Finally, owing to the reversibility of the process, gels can be restained after rapid visualization. For these reasons, negative staining with methyl trichloroacetate should become the method of choice for rapid and sensitive staining of proteins prior to further processing, including stable staining with silver ions.

Chemical Precipitation↗
PubMed1979

Low-dose electron image reconstruction of negatively stained contractile phage sheath from Bacillus subtilis (PBS-Z).

The structure of the contractile sheath of the defective phage from B. subtilis (PBS-Z) has been investigated by low-dose electron microscopy and image reconstruction. The extended and contracted sheath particles were imaged by means of two negative stains which consisted of uranyl- and phosphotungstate-containing solutions of a pH of 4.2 and 7.0 respectively. Images of identical parts of the same type of specimen were recorded at a total electron dose of 80 C/m2 (5 electrons/A2) and 4 x 10(3) C/m2 (250 electrons/A2). The low-dose reconstructions of the extended and contracted sheath structure in the two stains show good correspondence and made it possible to draw the following structural conclusions. The sheath protein in both types of structure has an elongated shape, and in both structures the long molecular axis lies in a plane perpendicular to the helical sheath axis. The orientation of the protein in the extended and contracted sheath is different; the long axes differ by about 35 degrees in orientation. The reconstructions did not permit conclusions about different conformational states of the protein in both structures. These data, together with the packing parameters of the protein subunits in the contractile sheath [1], form the complete structural analysis of this biological structure by electron microscopy. The radiation damage effects which have been monitored in analyzing image pairs to the full extent may be summarized as follows. (1) Diameters of the sheath structure increase, which indicate flattening. (2) There is no loss in resolution, and layerline altitudes of the Fourier-transformed images do not change. (3) Uranyl stain behaves differently compared to phosphotungstate. In both negative stains the structural noise level increases upon irradiation as follows from the increase in phase residuals of the digital layerline data. In uranyl-stained images also more aperiodic noise appears. (4) The Fourier amplitudes of the principal layerline maxima shift towards lower spatial frequencies; phases of corresponding maxima generally remain constant. This pattern is more pronounced in the extended sheath data; there is no rationale describing these positional shifts. Moreover, in the case of contracted sheath the amplitudes of Fourier components also change more in absolute value. Therefore the damage effects also seem to depend on the type of structure embedded in the stain. (5) In the reconstructed images these radiation effects create artificial stain-excluded volumes of a type and at a radius which depend on the stain and structure.

Bacillus subtilis↗
PubMed1982

Molecular sieve in rat glomerular basement membrane as revealed by negative staining.

Human and bovine glomerular basement membranes (GBM) were previously shown to be a three-dimensional molecular sieve composed of pores and strands by negative staining and electron microscopy. In this study, rat GBM were isolated under several different conditions to rule out morphological changes due to isolation procedures. Rat GBM isolated under different conditions all showed the same morphological features as bovine and human GBM. The strands forming the molecular sieve were almost equal in width, measuring approximately 3.1 +/- 0.8 nm. Pores were oval or polygonal. The size of pores varied a little averaging 4.4 +/- 1.0 nm in the long dimension and 3.0 +/- 0.6 nm in the short dimension. The average density of the pores was 16 +/- 2/1,000 nm2. Negative staining demonstrated pores in isolated and unfixed GBM, indicating that the function of GBM is mechanical filtration of macromolecules on the basis of size.

Animals↗
PubMed2000

Light atom derivatives of structure-preserving sugars are unconventional negative stains.

Although glucose and certain other sugars are known to greatly reduce distortion and denaturation of proteins during drying, use of this monosaccharide as an experimental negative stain does not permit imaging of lattice periodicities in test specimens of thin catalase crystals. However, the potassium and sodium salts of several forms of monophosphorylated glucose (200 mM), diphosphorylated glucose, monosulfated glucose, maltose-1-phosphate, and trehalose-6-phosphate, all dry into a glassy layer and scatter transmitted electrons sufficiently to show the 86 A major periods in catalase crystals. Glucose-6-phosphate provides sufficient image contrast at concentrations from 2 mM (=0.067%) to 500 mM (= 16.8%). Underfocusing increases visualization of the periodic lattice, indicating a large contribution of phase contrast to these images. Upon exposure to the electron beam, thicker regions of derivatized saccharides or pure glucose develop bubbling; this redistribution of dried stain largely can be precluded by imaging with low-dose exposures. Power spectra of images of catalase crystals contained within 200 mM disodium glucose-6-phosphate show that periodic information can be recorded to 21 A; some individual features of dipotassium glucose-6-phosphate distribution within the protein lattice have a measured width of around 5 A. The experimental results demonstrate that structure-preserving mono- and di-saccharides also serve successfully as negative stains after they are coupled to light atom scatterers.

Animals↗
PubMed2003

Use of cryo-negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage.

Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X-ray crystallography nor nuclear magnetic resonance. However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis. Indeed, cryo-electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining, which reduces the radiation damage while preserving a high signal-to-noise ratio. Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.

Bacteriophage T4↗
PubMed1994

Negatively-stained polysomes on rough microsome vesicles viewed by electron microscopy: further evidence regarding the orientation of attached ribosomes.

Rough microsomes, derived from rough endoplasmic reticulum of rat liver, were studied by electron microscopy after negative staining, to seek further information about the orientation of ribosomal small and large subunits in bound polysomes. Rough microsomal vesicles were fixed with 2% formaldehyde, centrifuged onto electron-microscopic grid membranes, and were then negatively-stained with 2% phosphotungstic acid. In these preparations, viewed with the electron microscope, flattened rough microsomal vesicles with bound polysomes were sometimes discernible, and the individual ribosomes in the polysomes occasionally showed small and large subunits. The small subunits were uniformly oriented toward the inside of the polysomal curve. The large and small subunits appeared to be alongside one another on the membrane, consistent with the orientation that has been described by Unwin and his co-workers. The boundary between the small and large subunits occurred at approximately the same level in the ribosome where inter-ribosomal strands have been described previously in surface views of bound polysomes in positively-stained electron-microscopic tissue sections. This further confirms the identity of the strands as messenger RNA.

Animals↗
PubMed1983

In situ electron microscopic observation of negatively stained tissue culture cells contaminated with mycoplasmas.

A simple, fast, and in situ method of detecting the inapparent infection of cultured cells with mycoplasmas is reported. Animal cells grown on Formvar-coated electron microscopic grids were directly fixed with glutaraldehyde, negatively stained with phosphotungstic acid and examined by transmission electron microscopy. Cells contaminated with mycoplasmas could be discriminated from uncontaminated cells. The micro-organisms in the negatively stained preparations corresponded with those revealed by thin sectioning, and the distribution of mycoplasmas in cultured cells coincided with those revealed by the Hoechst staining method. Most of the highly resolved mycoplasmas were polymorphic, and closely associated with host cells; often more than 500 organisms per host cell were seen.

Animals↗
PubMed1984

Negative staining method with nigrosin for the detection of cryptosporidial oocysts: a comparative study.

A comparison was made between two methods for the detection of cryptosporidial oocysts in human faecal sediments of formalin-ether concentrates: a cover-slipped, wet method (containing Gram's iodine) and an air-dried, negative staining method using nigrosin solution. A modified Ziehl-Neelsen technique was used as a reference method. The negative staining method with nigrosin gave a positive diagnosis more often than the cover-slipped wet method. A comparison of the nigrosin method with the modified Ziehl-Neelsen method gave almost identical results. Restaining the nigrosin slides with the modified Ziehl-Neelsen technique showed that the round, refractile bodies were cryptosporidial occysts.

Aniline Compounds↗
PubMed1973

Ultrastructural properties of the extra membranes of Escherichia coli O111a as revealed by freeze-fracturing and negative-staining techniques.

Escherichia coli O111a is a thermosensitive strain which, when grown at 40 C, accumulates large quantities of intracellular membranes. The ultrastructure of these membranes in cells which have been chemically fixed, embedded, and examined as thin sections has been compared with that of membranes in cells negatively stained or freeze-fractured. Results indicate that the extra membranes are present in the three types of preparations examined and, therefore, clearly are not artifacts of chemical fixation. Negative staining has proved also to be a valuable tool as a rapid means of monitoring cells for the accumulation of large amounts of extra membranes. Also, examination of thin sections has shown that distinct continuities between the plasma membrane and the extra membranes exist. In general, membrane surfaces in freeze-fractured cells containing extra membranes appear smooth and lack the particles associated with the plasma membranes of many cells.

Cell Membrane↗