Emerg Infect Dis [serial online] Volume 9, Number 3, March 2003

Electron Microscopy for Rapid Diagnosis of Emerging Infectious Agents

Paul R. Hazelton* and Hans R. Gelderblom**
*University of Manitoba, Winnipeg, MB, Canada; and **Robert Koch-Institut, Berlin, Germany

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

... Rapid immunologic methods that improve sensitivity [of diagnostic electron microscopy] when searching for unknown agents include both of which may use either pooled human immunoglobulins (HuIgG) or specific antibodies. HuIgG may be obtained from most immunologic suppliers or hospital pharmacies.

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


SIA uses immunoaggregation to identify antigens.

Diluent diffuses into the agar while antibody diffuses into the suspension and antigen:antibody complexes form, which then adsorb to the grid as diluent volume is reduced (Figure 4B)

Figure 4.

Figure 4. Two-step staining preparation of suspension samples.

Negative Staining

Biologic structures, because of low mass density, interact weakly with electrons used for imaging, and therefore, show little contrast or detail. Several ways exist to generate sufficient image contrast and resolution; the most versatile is positive and negative staining with heavy metal ions, e.g., lead, tungsten, and uranium ions.


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

Biel SS, Gelderblom HR. Electron microscopy of viruses. In: Cann A, editor. Virus cell cultureča practical approach. Oxford: Oxford University Press; 1999. p. 111?47.

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.
 



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