Negative staining

Tuscheverfahren, n. (Micros.) India ink method, negative staining. 

FIG. 5 Electron micrographs of negatively stained preparations of S-layer self-assembly products, (a) flat sheet (bar, 100 nm), (b) open-ended cylinder (bar, 200 nm). 

Electron transmission micrographs of negatively stained crystalline precipitates revealed a highly ordered three-dimensional crystalline array with intermolecular distances in good agreement with the predesigned diamond-like model. 

Unlike heliantholysin and congeners, the toxicity of metridiolysin is not prevented by sphingomyelin, but is inhibited by cholesterol in low concentration, as well as by certain related sterols (23). In addition, metridiolysin is activated by thiols such as dithiothreitol, and is reversibly inactivated by compounds having an affinity for SH-groups, such as p-hydroxy-mercuribenzoate. A third notable feature is that the action of metridiolysin on membranes involves, or is associated with, the formation of 33 nm rings demonstrable by electron microscopy of negatively stained preparations. 

Fig 2 Immunogold negative staining, with a monoclonal antibody (JIM 5 (13)) that recognises a relatively unesterified pectic epitope, of rhamnogalacturonans extracted from onion cell walls. Arrows indicate 5 nm colloidal gold particles. Scale bar represents 200nm. 

Fig. 1. Negative staining by phosphotungstic acid of Na,K-ATPase purified in membrane-bound form. The membrane surfaces are covered by particles arranged in clusters between smooth areas. From [2] procedure as described by Deguchi et al. [30].

More than half of the total mass of the ATPase molecule is exposed on the cytoplasmic surface of the membrane, forming the 40-A x 60-A particles seen by negative staining electron microscopy [88 93]. 

The dimer chains of Ca -ATPase can also be observed by freeze-fracture electron microscopy [119,165,166,172-174], forming regular arrays of oblique parallel ridges on the concave P fracture faces of the membrane, with complementary grooves or furrows on the convex E fracture faces. Resolution of the surface projections of individual Ca -ATPase molecules within the crystalline arrays has also been achieved on freeze-dried rotary shadowed preparations of vanadate treated rabbit sarcoplasmic reticulum [163,166,173,175]. The unit cell dimensions derived from these preparations are a = 6.5 nm b = 10.7 nm and 7 = 85.5° [175], in reasonable agreement with earlier estimates on negatively stained preparations [88]. 

Image reconstruction in three dimensions from negatively stained and frozen hydrated crystals... 

The cytoplasmic domains reconstructed from negatively stained [90] and from frozen-hydrated samples [91,177] have similar shapes. Both include the protruding lobe and the bridge region that links the Ca " -ATPase molecules into dimers. The intramembranous peptide domains of the two ATPase molecules which make up a dimer spread apart as they pass through the bilayer toward the luminal side of the membrane, establishing contacts with the Ca -ATPase molecules in the neighboring dimer chains. The lateral association of dimer chains into extended crystal lattice is... 

Analysis of the lanthanide-induced crystalline arrays by negative staining (Fig. 5) or freeze-fracture electron microscopy reveals obliquely oriented rows of particles, corresponding to individual Ca -ATPase molecules [119]. The unit cell dimensions for the gadolinium-induced Ca -ATPase crystals are a = 6. l A, b = 54.4 A and y = 111°. Similar cell constants were obtained for the crystals induced by lanthanum, praseodymium and calcium. The unit cell dimensions of the Ei crystals are consistent with a single Ca -ATPase monomer per unit cell. The space group of the Eptype crystals is PI [119], while that of the E2 crystals is P2 [88,90]. 

Two distinct patterns of repeats were observed by electron microscopy of sectioned, negatively stained, frozen-hyd rated, or freeze-fractured specimens of Ca -ATPase crystals that represent different projections of the same structure... 

Fig. 7. Projection view of negatively stained Ca -ATPase crystals in sarcoplasmic reticulum solubilized with Ci2Eg (2 mg/mg protein) in the standard crystallization medium. The prominent large spacing is the half-period of the a cell dimension. Striations oblique to this direction are the (1,1) and the (-1,1) periodicities. Magnification, x 308 000. From Taylor et al. [156].

The projection map of the unstained frozen-hydrated three-dimensional crystals was compared with the map of negatively stained crystals [141,142] to define the densities associated with the intramembranous domains. The ten transmembrane helices were predicted to be arranged in two crescent shaped rows that provide intramembranous contacts between ATPase molecules. The scheme is entirely speculative and with few exceptions there is no clear relationship between the density contours seen in the map and the proposed transmembrane helices. 

J du Plessis, LR Tiedt, AF Kotze, CJ van Wyk, C Ackerman. A transmission electron microscope method for determination of droplet size in parenteral fat emulsions using negative staining. Int J Pharm 46, 1988. 

Figure 6.4 TEM image of the dispersion of nanoparticles obtained after evaporation of the solvent of a nanoemulsion with an O/S of 70 30 and a water content of 90wt% and negative staining with a phosphotungstic acid solution. Reproduced with permission from [54].

Measurements show some variation depending upon the staining solution used and the method of application. In dried and fixed smears, the cell wall and slime layer do not stain with weakly staining dyes such as methylene blue but do stain with the intensely staining pararosaniline, new fuchsin, crystal violet, and methyl violet. The great majority of bacteria have been measured in fixed and stained preparations. In some instances dried, negatively stained smears have been used. Therefore, the method employed should be specified when measurements of bacteria are reported otherwise the results will be of doubtful v alue. 

Figure 5.22 Electron micrograph by negative staining of a lambda bacteriophage particle.

Figure 6.13 Electron micrograph of human spleen ferritin viewed at a magnification of x 170 000 after negative staining with uranyl acetate. Both the darkly coloured iron-rich cores and the clear-coloured protein shells are clearly visible. (From Crichton, 1991.)...

Figure 5.29 Negative-stained electron micrograph of DCs,9 PC tubule from 5-mg/ml sample in methanol-water (85 15). Solvent conditions and lipid concentration were adjusted to obtain sample of two-bilayer tubules as shown in enlargement of tubule edge in bottom panel. Bar = 200 nm (top) 50 nm (bottom). Reprinted with permission from Ref. 125. Copyright 1998 by the American Chemical Society.

Figure 5.38 (a) Negative-stained transmission electron micrograph of nanotubules formed from equimolar mixture of DCg PC and DNPC (2 mM total lipid concentration) stored at 4°C just prior to deposition, (b) Freeze-fracture electron micrograph of twisted ribbons at 27°C. Bars = 100 nm. Reprinted with permission from Ref. 153. Copyright 2001 by the American Chemical Society. 

Nearfield scanning optical Determine single molecules on surfaces Negative staining Useful for detergent-extracted cytoskeletons, membrane fractions, organelles... 

Hayat MA. Negative Staining, McGraw Hill, New York, 1990. 

Harris R, Home R. Negative staining, in Electron Microscopy in Biology—A Practical Approach (Harris JR, ed.), IRL Press, Oxford, UK, 1991, pp. 203-228. 

Subsequent to possible solubilization of membrane-bound proteins, solubilization must be verified. The criteria listed in Table 2 are relevant in assessing whether solubilization has been accomplished. To ascertain whether the solubilized protein has retained biological activity, membrane reconstitution (28) is attempted subsequent to detergent removal (24). Reconstitution is often visualized by electron microscopy employing either negative staining or freeze fracture. 

I. Negative Staining. This technique differs frompositive staining in that heavy metals surround the specimen leaving the latter unstained... 

Negative stains a Concentration and useful pH range Application(s) Reference(s)... 

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