Unconventional negative stains: Heavy metals are not required for negative staining

Subsequent to its introduction a few years ago by Mizuhira and Fukaesaku (1) the practice of using tannic acid (TA) in glutaraldehyde (GA) fixatives followed by heavy metal postfixation has become a standard technique for revealing the substructure of microtubules. We showed in a previous report that a reaction similar to that between TA and tubulin also occurs between TA and actin. Actin filaments of striated muscle appear negatively stained in situafter GA-TA fixation and osmium postfixation (2). That result suggested that GA-TA fixatives might be applied to studies of actin-like filaments in non-muscle cells. However, in light of the results of Szamier et at(3) showing that naked (not protected by tropomyosin) F-actin is destroyed by osmium, it was questionable whether it was possible to preserve and negatively stain naked F-actin, as well as actin-like microfilaments, with GA-TA fixatives. Also, we did not know if osmium must be used as a postfixative or whether other heavy metals could be substituted for it.

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The Champagne Artifact in Negative Staining TEM

Microscopy Today ◽

10.1017/s1551929500069704 ◽

1998 ◽

Vol 6 (10) ◽

pp. 24-25

Author(s):

John J. Bozzola

Keyword(s):

Heavy Metals ◽

Negative Staining ◽

Extensive Literature ◽

Basic Procedure ◽

Established Procedure ◽

Amorphous Glass ◽

Carbon Backing

Negative staining is a simple yet extremely useful procedure for examining nanometer-sized specimens such as intact microorganisms (viruses and some bacteria), subcellular components, and even nonbidogical particulates. It is a well established procedure, with extensive literature in many disciplines (Hayat and Miller, 1990). Although numerous variations exist, the basic procedure involves placing the specimen and stain onto a grid containing a substrate - usually plastic with or without a carbon backing. The stains consist of salts of heavy metals such as tungsten, uranium, or molybdenum which surround and often penetrate the specimen. Afier drying into an amorphous, glass-like background, the stains provide contrast based upon differential thickness.

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Polymer Morphology Revealed by Staining Techniques

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098204 ◽

1981 ◽

Vol 39 ◽

pp. 340-341

Author(s):

A. C. Reimschuessel ◽

V. Kramer

Keyword(s):

Polymer Melt ◽

Nylon 6 ◽

Morphological Features ◽

Negative Staining ◽

Polymer Morphology ◽

Crystallizable Polymer ◽

Staining Techniques ◽

Long Chain

Staining techniques can be used for either the identification of different polymers or for the differentiation of specific morphological domains within a given polymer. To reveal morphological features in nylon 6, we choose a technique based upon diffusion of the staining agent into accessible regions of the polymer.When a crystallizable polymer - such as nylon 6 - is cooled from the melt, lamellae form by chainfolding of the crystallizing long chain macromolecules. The regions between adjacent lamellae represent the less ordered amorphous domains into which stain can diffuse. In this process the lamellae will be “outlined” by the dense stain, giving rise to contrast comparable to that obtained by “negative” staining techniques.If the cooling of the polymer melt proceeds relatively slowly - as in molding operations - the lamellae are usually arranged in a radial manner. This morphology is referred to as spherulitic.

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New ultrastructural findings about Borrelia burgdorferi by cryofixation, negative staining, thin sectioning and scanning techniques

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160467 ◽

1990 ◽

Vol 48 (3) ◽

pp. 582-583

Author(s):

S. F. Hayes ◽

M. D. Corwin ◽

T. G. Schwan ◽

D. W. Dorward ◽

W. Burgdorfer

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Structural Characterization ◽

Negative Staining ◽

Low Speed ◽

Thin Sectioning ◽

Ultrastructural Findings

Characterization of Borrelia burgdorferi strains by means of negative staining EM has become an integral part of many studies related to the biology of the Lyme disease organism. However, relying solely upon negative staining to compare new isolates with prototype B31 or other borreliae is often unsatisfactory. To obtain more satisfactory results, we have relied upon a correlative approach encompassing a variety EM techniques, i.e., scanning for topographical features and cryotomy, negative staining and thin sectioning to provide a more complete structural characterization of B. burgdorferi.For characterization, isolates of B. burgdorferi were cultured in BSK II media from which they were removed by low speed centrifugation. The sedimented borrelia were carefully resuspended in stabilizing buffer so as to preserve their features for scanning and negative staining. Alternatively, others were prepared for conventional thin sectioning and for cryotomy using modified procedures. For thin sectioning, the fixative described by Ito, et al.

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Sampling and analysis of the air-water interface with SEM and EDS of microlayers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157000 ◽

1989 ◽

Vol 47 ◽

pp. 1004-1005

Author(s):

Randall W. Smith ◽

John Dash

Keyword(s):

Heavy Metals ◽

Surface Microlayer ◽

Surface Films ◽

Water Interface ◽

Physical Processes ◽

Infrared Spectroscopic Analysis ◽

Eds Analysis ◽

Air Water Interface ◽

Significant Area ◽

Bulk Chemical

The structure of the air-water interface forms a boundary layer that involves biological ,chemical geological and physical processes in its formation. Freshwater and sea surface microlayers form at the air-water interface and include a diverse assemblage of organic matter, detritus, microorganisms, plankton and heavy metals. The sampling of microlayers and the examination of components is presently a significant area of study because of the input of anthropogenic materials and their accumulation at the air-water interface. The neustonic organisms present in this environment may be sensitive to the toxic components of these inputs. Hardy reports that over 20 different methods have been developed for sampling of microlayers, primarily for bulk chemical analysis. We report here the examination of microlayer films for the documentation of structure and composition.Baier and Gucinski reported the use of Langmuir-Blogett films obtained on germanium prisms for infrared spectroscopic analysis (IR-ATR) of components. The sampling of microlayers has been done by collecting fi1ms on glass plates and teflon drums, We found that microlayers could be collected on 11 mm glass cover slips by pulling a Langmuir-Blogett film from a surface microlayer. Comparative collections were made on methylcel1ulose filter pads. The films could be air-dried or preserved in Lugol's Iodine Several slicks or surface films were sampled in September, 1987 in Chesapeake Bay, Maryland and in August, 1988 in Sequim Bay, Washington, For glass coverslips the films were air-dried, mounted on SEM pegs, ringed with colloidal silver, and sputter coated with Au-Pd, The Langmuir-Blogett film technique maintained the structure of the microlayer intact for examination, SEM observation and EDS analysis were then used to determine organisms and relative concentrations of heavy metals, using a Link AN 10000 EDS system with an ISI SS40 SEM unit. Typical heavy microlayer films are shown in Figure 3.

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