Negative staining is an excellent method for the preservation and contrast enhancement of objects ranging in size from macromolecules to entire organisms, such as bacteria. This method is outstanding among electron microscopy preparation methods for its easy and rapid procedure (1). In addition to increasing the contrast, negative stain (i) also protects the specimen from distortion caused by dehydration as the stain displaces the water surrounding the specimen, and (ii) stabilizes against collapse in the vacuum of the electron microscope. However, care must be observed when choosing a stain since the preparation protocol and choice of stain can produce different results. Furthermore, the wrong stain can disorder the structure.
Staining is achieved by using heavy metal salts which have the property of drying from an aqueous solution to an amorphous "glass." The metal salt solution is able to occupy the hydrated regions in and around the particle, and as the solution dries it forms an amorphous electron-dense replica of the particle. The image formed is light in areas occupied by biological structure (said to be stain-excluding) and dark in areas occupied by stain. Hence, the technique is called "negative" staining, since the image has the appearance of a photographic negative. The degree of permeation for a particular stain may be influenced by local charge distributions and variations in the degree of hydrophilicity of the specimen (2). A number of heavy metal stains have been employed, but uranyl acetate, phosphotungstic acid, and ammonium molybdate are the most commonly used because they exhibit excellent structural detail. The high contrast of negatively stained preparations even permits the visualization of individual protein molecules (Fig. 1) that are relatively small (>20 Kdal). Negative stain embedment also reduces the problem of radiation damage, since the negative stain replica, which is more resistant to electron radiation, supports the biological specimen. However, at higher electron doses, stain redistribution can cause a loss of resolution (3).
Figure 1. Molecules of E. coli glutamine synthetase negatively stained with uranyl acetate. This enzyme is composed of 12 identical subunits of 50,000 MW each arranged at the vertices of two eclipsed hexagons to produce a structure with 622 symmetry with dimensions of 145 A by 95 A. Notice the well-defined features of isolated molecules many of which show six-fold symmetry or two-fold symmetry depending on orientation. Glutamine synthetase molecules have a tendency to aggregate along the six-fold axes forming stacks of molecules which can be seen in this image.
Negative stain is most useful for revealing (i) the molecular envelope in projection and in 3-D reconstruction, (ii) the orientation of resolvable subunits in complexes, and (iii) the interactions and interfaces between molecules in assemblies and crystals. One of the downfalls of negative staining is that the resolution limit for most specimens is ~1.5 nm, even when the structure possesses higher order. This limit is probably set by the grain size of the stain. However, there have been reports of images of negatively stained specimens with a resolution of 1.0 nm or better. This increased resolution is usually accomplished at cryotemperatures and with mixtures of negative stain and sugars or aurothioglucose (4-6), although a resolution of 0.8 nm has also been reported in negative stain without adding sugar or resorting to cryotemperature (7). In limited cases, negative stain can produce higher resolution than vitreous ice embedment, as shown for crystals of cytochrome c oxidase (6) in which 0.7 nm resolution was achieved in negative stain, while only 1.0 nm resolution was achieved with ice-embedded crystals. The lower resolution in ice may be ascribed to beam-induced specimen movement or specimen charging. The ease of negative stain makes it useful for surveying crystallization conditions and the degree of order of newly grown crystals, which may be used to ascertain if a higher resolution medium is warranted (8). When judging between the relative merits of negative stain and of a higher resolution medium, at times it is important to ask, "What level of structural detail will answer the research question?" For example, negative stain was chosen over ice embedment for crystals of biochemically split gap junctions as the best medium to enhance the contrast in order to visualize the extracellular domains that had been previously unseen (9).
