Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bleck, C K E; Merz, A; Gutierrez, M G; Walther, P; Dubochet, J; Zuber, Benoît; Griffiths, G (2010). Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis. Journal of microscopy, 237(1), pp. 23-38. Oxford: Wiley-Blackwell 10.1111/j.1365-2818.2009.03299.x

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Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Anatomy

UniBE Contributor:

Zuber, Benoît

Subjects:

600 Technology > 610 Medicine & health

ISSN:

0022-2720

Publisher:

Wiley-Blackwell

Language:

English

Submitter:

Benoît Zuber

Date Deposited:

04 Oct 2013 14:15

Last Modified:

05 Aug 2016 12:55

Publisher DOI:

10.1111/j.1365-2818.2009.03299.x

PubMed ID:

20055916

Web of Science ID:

000272659400004

BORIS DOI:

10.7892/boris.4094

URI:

https://boris.unibe.ch/id/eprint/4094 (FactScience: 208215)

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