Electron Microscopy for Rapid Diagnosis of Emerging Infectious Agents

Diagnostic electron microscopy has two advantages over enzyme-linked immunosorbent assay and nucleic acid amplification tests. After a simple and fast negative stain preparation, the undirected, "open view" of electron microscopy allows rapid morphologic identification and differential diagnosis of different agents contained in the specimen ...

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Specimen Preparation

• solid-phase immunoelectron microscopy (SPIEM) (Lewis DC) and
• serum in agar (SIA) (Anderson N, Doane FW),

SPIEM concentrates antigens on the grid by immune capture, thereby improving the probability of observing an etiologic agent.

• The coated surface of a grid is floated on a drop of pooled HuIgG (100 µg/mL and 20 µg/mL in phosphate-buffered saline [PBS] B) or antiserum (1/100 and 1/500 in PBS) for 10 min,
• washed on 6 sequential drops PBS, and
• floated on the specimen for 30?60 min at 37°C.
• The sample may be stabilized after SPIEM with 0.1% glutaraldehyde to ensure tight binding of the captured antigens,
• washed on 6 drops of PBS,
• negative stained, and examined (Lewis DC).

SIA uses immunoaggregation to identify antigens.

• In addition, type-specific antisera may be used in SIA to serotype the agent present. Antibody (1/100 for antisera and 100 µg/mL for HuIgG) is prepared in cooled 1% agar.
• A grid is placed on the solidified agar, and
• a drop of sample placed over the grid.

Negative Staining

• Positive staining depends on chemical reactivity with the components of the object and involves fixation, postfixation, embedding in resins, ultrathin sectioning, and multiple staining incubations. These procedures may take 4?5 days before a sample is ready for examination. Rapid embedding protocols can reduce the time to approximately 1 day but with a loss in specimen quality (Biel SS, Gelderblom HR).
• In contrast, negative staining is simple, rapid, and well suited for examination of small particulate suspensions.
• A coated grid with sample adsorbed to the surface is floated on a drop of negative stain for 0.5 ... 2 min,
• excess stain wicked away with a piece of filter paper,
• air dried for 1 ... 3 min, and
examined by electron microscopy (Figure 4D). Structures on the grid are surrounded and stabilized by the drying stain. Thus, they appear as transparent, highly detailed negative images within a dark halo of stain (Figure 5B).

Figure 5. Comparison of herpesvirus appearance after positive and negative stain electron microscopic. A. Positive staining. Samples undergo a lengthy process of fixation, incubation with heavy metal ions (osmium, uranyl), dehydration, embedment, ultrathin sectioning, and staining. Chemical moieties in the object show differential affinities for the heavy metal stains, resulting in a clear outline of the viral bilayer envelope, viral envelope proteins, nucleocapsid, and the dense nucleic acid containing core. B. Negative staining. After a brief fixation, samples are mounted directly on electron microscopic grids and stained as in Figure 4. The electron-dense stain (phosphotungstic acid [phosphotungstic acid], uranyl acetate, and the like) penetrates the virion and embeds the particle in a matrix of stain. Due to density differences between the stain and weakly scattering biological components of the virion, the virion appears as a transparent and detailed reverse (negative) image. Penetration of stain into the nucleocapsid provides a dense core with the crenellated appearance presented by the central channel of capsomers on the nucleocapsid surface. Viral surface proteins appear as projections from the labile envelope. Phosphotungstic acid stained. Bar = 100 nm.

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References

Lewis DC. Three serotypes of Norwalk-like virus demonstrated by solid-phase immune electron microscopy. J Med Virol 1990;30:77?81.

Anderson N, Doane FW. Specific identification of enteroviruses by immuno-electron microscopy using a serum-in-agar diffusion method. Can J Microbiol 1973;19:585?9.