Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism-related micro-organisms

Abstract

Aims: To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism. Methods and Results: Suspensions of vaccinia poxvirus and endospores of Bacillus subtilis were used at defined concentrations as a model for poxviruses and spores of anthrax (Bacillus anthracis), both of which are pathogens that could be used for bioterrorist attacks. Negative staining electron microscopy was performed directly or after sedimentation of these suspensions on to the sample supports using airfuge ultracentrifugation. For both virus and spores, the detection limit using direct adsorption of a 10‐μl sample volume onto the sample support was 106 particles per ml. Using airfuge ultracentrifugation with a sample volume of 80 μl, the detection limit could be reduced to 105 particles per ml for spores and to 5 × 104 particles per ml for poxviruses. The influence on particle detection of incubation time, washing and adsorption procedures was investigated. Conclusions: The reproducibility and sensitivity of the method were acceptable, particularly considering the small sample volume and low particle number applied onto the sample support. Significance and Impact of the Study: Diagnostic negative staining electron microscopy is used for the diagnosis of pathogens in emergency situations because it allows a rapid examination of all particulate matter down to the nanometre scale. This study provides precise detection limit for the method, an important factor for the validation and improvement of the technique.

Rapid and reliable detection of bacterial endospores in environmental samples by diagnostic electron microscopy combined with X-ray microanalysis

“…Spores were adsorbed at the sample support, washed and stained by heavy metals according to evaluated protocols (Laue and Bannert, 2010). As a standard sample support, copper grids (400 or 300 mesh; AGAR Scientific) that have been coated with plastic (pioloform or formvar) and carbon (to provide a better heat distribution and mechanical stability) were used.…”

“…As a standard sample support, copper grids (400 or 300 mesh; AGAR Scientific) that have been coated with plastic (pioloform or formvar) and carbon (to provide a better heat distribution and mechanical stability) were used. To make the surface more hydrophilic and sticky, the carbon reinforced plastic film was covered by the cationic stain alcian blue (1% in 1% acetic acid, 10 min; Laue and Bannert, 2010). After washing of the grids on four droplets of double distilled water, 10 μl of the spore suspension was dispensed directly onto the coated grid surface.…”

“…Airfuge (Beckman) sedimentation of samples directly onto the surface of the grid was performed as described by Laue and Bannert (2010). Briefly, alcian blue coated grids were fitted into particular grid adaptors (Type S⁄N 833101329, Beckman; No.…”

“…Ten samples per defined spore concentration were systematically inspected with a scanning electron microscope (TM-1000) by screening 22 meshes using a regular pattern which involves all regions of the sample ( Laue and Bannert, 2010). Particles revealing the morphology of spores (size, shape, and density) were analysed by X-ray microanalysis to check the X-ray spectrum for the typical signature of spores.…”

“…The detection of spores by negative staining electron microscopy is generally possible (Laue and Bannert, 2010). Spores that possess a typical exosporium, like spores of anthrax, can be distinguished from spores which are lacking this particular outer coat.…”