Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis.
Details
Serval ID
serval:BIB_77FE215A1BF7
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis.
Journal
Journal of Microscopy
ISSN
1365-2818 (Electronic)
ISSN-L
0022-2720
Publication state
Published
Issued date
2010
Peer-reviewed
Oui
Volume
237
Number
1
Pages
23-38
Language
english
Abstract
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.
Keywords
Artifacts, Cell Wall/ultrastructure, Cryoelectron Microscopy/methods, Cytoplasm/ultrastructure, DNA, Bacterial/ultrastructure, Epoxy Resins, Freeze Substitution/methods, Microscopy, Electron, Transmission/methods, Microtomy, Mycobacterium smegmatis/radiation effects, Mycobacterium smegmatis/ultrastructure, Temperature, Tissue Fixation/methods, Ultraviolet Rays
Pubmed
Web of science
Create date
22/03/2011 10:32
Last modification date
20/08/2019 15:34