Cryo-negative staining

Abstract A procedure is presented for the preparation of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we termcryo-negative staining. The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films across the holes, which are routinely thiner than the aqueous film produced by conventional negative staining on a continuous carbon layer. Because of this, a higher than usual concentration of negative stain (ca. 16% rather than 2%) is required for cryo-negative staining in order to produce an optimal image contrast. The maintenance of the hydrated state, the absence of adsorption to a carbon film and associate…

Amylopectin: a major component of the residual body inCryptosporidium parvumoocysts

Amylopectin is used for carbohydrate storage in different life-stages of a number of apicomplexan parasites. We have performed an ultrastructural analysis of amylopectin granules from the oocyst residual body and sporozoites ofCryptosporidium parvum. Amylopectin granules were studiedin situand after isolation from ‘French’ press disrupted parasites, by conventional transmission electron microscopy (TEM) of sectioned oocysts and various negative staining and cryoelectron microscopy techniques. Within the membrane-enclosed oocyst residuum large amylopectin granules (0·1–0·3 μm) can be found besides a characteristic large lipid body and a crystalline protein inclusion. Smaller granules were de…

Formation of two-dimensional crystals of icosahedral RNA viruses.

International audience; The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A…

Transmission electron microscopical studies on some haemolymph proteins from the marine polychaete Nereis virens.

Abstract The hexagonal bilayer haemoglobin molecule from Nereis virens has been investigated in a comparative study using several different negative stain electron microscopical specimen preparations (i.e. by conventional adsorption to continuous carbon support films, by the negative staining-carbon film technique and by negative staining across the holes of holey carbon support films with air-drying and rapid freezing/cryo-negative staining). The benefits and limitations of these different approaches are indicated, with the overall conclusion that negative staining with ammonium molybdate across holes creates the best possibilities for molecular imaging, and also has the potential for the …

Cholesterol-Streptolysin O Interaction: An EM Study of Wild-Type and Mutant Streptolysin O

We present transmission electron microscopical data from negatively stained specimens of cholesterol following interaction with the thiol-activated bacterial toxin streptolysin O (SLO) (wild-type and a number of cysteine substitution mutants), with and without chemical modification of the cysteine residues. Two experimental systems were used, one with an aqueous suspension of cholesterol microcrystals and the other with immobilized thin planar cholesterol crystals attached to a carbon film. In both systems the wild-type SLO and two cytolytically active mutants, Cys 530 --Ala (C530A) and Ser 101 --Cys (S101C), readily generated the characteristic SLO arc- and ring-like oligomers on the surfa…

Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile apicomplexan organelle.

From an EM study of thin sections, the rod-like microneme organelles within conventionally glutaraldehyde fixed Cryptosporidium parvum sporozoites have been shown to undergo a shape change to a more spherical structure when the sporozoites age in vitro for a period of approximately 12 to 24 h. This correlates with the shape change of intact sporozoites, from motile hence viable thin banana-shaped cells to swollen pear-shaped cells, shown by differential interference contrast light microscopy of unstained unfixed and glutaraldehyde-fixed samples, as well as by thin section EM of fixed sporozoites. From negatively stained EM specimens of unfixed and fixed sporozoites the cellular shape change…

Preparation of Thin-Film Frozen-Hydrated/ Vitrified Biological Specimens for Cryoelectron Microscopy

Keyhole Limpet Hemocyanin (KLH): Slow In Vitro Reassociation of KLH1 and KLH2 from Immucothel®

Abstract Following our in vitro reassociation of keyhole limpet hemocyanin subunits in the presence of high concentrations (100 mM each) of calcium and magnesium chloride (Harris et al., 1997a, Micron 28, 31–41; 1997b, Micron 28, 43–56), we have now extended our investigations by using a buffer system containing a lower concentration of the two divalent cations (10 mM each). Reassociation of mixed KLH subunits present in the commercially available product Immucothel® was performed using a standardized buffer solution containing 50 mM Tris–HCl, 150 mM NaCl, 10 mM CaCl2 and 10 mM MgCl2 (pH 7.4) over a minimum period of one week, at 4°C. This solution was selected as being close to our KLH sta…