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The scanning electron microscope in microbiology and diagnosis of infectious disease

Golding, Christine G., Lamboo, Lindsey L., Beniac, Daniel R., Booth, Timothy F.·2016·Scientific Reports
DOI 10.1038/srep26516PMC PMC4876401
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Abstract

Despite being an excellent tool for investigating ultrastructure, scanning electron microscopy (SEM) is less frequently used than transmission electron microscopy for microbes such as viruses or bacteria. Here we describe rapid methods that allow SEM imaging of fully hydrated, unfixed microbes without using conventional sample preparation methods. We demonstrate improved ultrastructural preservation, with greatly reduced dehydration and shrinkage, for specimens including bacteria and viruses such as Ebola virus using infiltration with ionic liquid on conducting filter substrates for SEM.

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Figure 1 Comparison of conventional sputter coating SEM sample preparation methods (panels on the left-hand side) with ionic liquid treatment (centre panels) and conventional TEM (panels on the right-hand side) for the observation of microbes: (a) Leptospira biflexa, (b) Salmonella Senftenberg, (c) vaccinia, and (d) Ebola virus. The SEM images in the left-hand side panels were of specimens that were sputter coated with gold, on plain uncoated filters. Images in the centre panels were of specimens treated with ionic liquid after deposition on pre-coated aluminum filters. On the right-hand side, TEM images of similar specimens prepared using methylamine tungstate negative staining.
Figure 1 Comparison of conventional sputter coating SEM sample preparation methods (panels on the left-hand side) with ionic liquid treatment (centre panels) and conventional TEM (panels on the right-hand side) for the observation of microbes: (a) Leptospira biflexa, (b) Salmonella Senftenberg, (c) vaccinia, and (d) Ebola virus. The SEM images in the left-hand side panels were of specimens that were sputter coated with gold, on plain uncoated filters. Images in the centre panels were of specimens treated with ionic liquid after deposition on pre-coated aluminum filters. On the right-hand side, TEM images of similar specimens prepared using methylamine tungstate negative staining.CC BY · PubMed Central
Figure 2 Preparation of biological samples for SEM. (a) The components of the filter unit are shown before assembly. The inset SEM image shows a gold coated filter at high magnification before a specimen is applied. Note that the filter is clean and the pores are clearly evident. (b) The filter unit is shown after assembly, and in use with a syringe pump in a biosafety cabinet (c,d). The blue arrow in (d) points to the filter unit. (e) Images of filters that have had metal evaporated on them. The thickness of the Al is 18 nm, and 9 nm, and the Au is 27 nm and 9 nm thick. For the filter with both metals the thickness of the Al and Au are 18 nm and 27 nm, respectively. (f) SEM and (g) the corresponding elemental map generated by X-ray microanalysis of a region similar to the one highlighted by the dotted rectangle in (e). (h–k) SEM images of Salmonella stained with ionic liquid illustrating the effect of different metal types, and thickness of metal evaporated on the final images recorded (h Al 9 nm, i Au 9 nm, j Al 18 nm, k Au 27 nm). Red arrows indicate flagellae.
Figure 2 Preparation of biological samples for SEM. (a) The components of the filter unit are shown before assembly. The inset SEM image shows a gold coated filter at high magnification before a specimen is applied. Note that the filter is clean and the pores are clearly evident. (b) The filter unit is shown after assembly, and in use with a syringe pump in a biosafety cabinet (c,d). The blue arrow in (d) points to the filter unit. (e) Images of filters that have had metal evaporated on them. The thickness of the Al is 18 nm, and 9 nm, and the Au is 27 nm and 9 nm thick. For the filter with both metals the thickness of the Al and Au are 18 nm and 27 nm, respectively. (f) SEM and (g) the corresponding elemental map generated by X-ray microanalysis of a region similar to the one highlighted by the dotted rectangle in (e). (h–k) SEM images of Salmonella stained with ionic liquid illustrating the effect of different metal types, and thickness of metal evaporated on the final images recorded (h Al 9 nm, i Au 9 nm, j Al 18 nm, k Au 27 nm). Red arrows indicate flagellae.CC BY · PubMed Central

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Metadata

Authors
Golding, C. G., Lamboo, L. L., Beniac, D. R., & Booth, T. F.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2016-05-23
Volume
6
Issue
1
ISSN
2045-2322
DOI
10.1038/srep26516
PMC
PMC4876401
Cited by
236
References
0
Language
en
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