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Journal of Bacteriology, October 2003, p. 6112-6118, Vol. 185, No. 20
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.20.6112-6118.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cryo-Transmission Electron Microscopy of Frozen-Hydrated Sections of Escherichia coli and Pseudomonas aeruginosa

Valério R. F. Matias,1 Ashraf Al-Amoudi,2 Jacques Dubochet,2 and Terry J. Beveridge1*

Biophysics Interdepartmental Group and Department of Microbiology, College of Biological Science, University of Guelph, Guelph, Ontario, Canada,1 Laboratoire d'Analyse Ultrastructurale, BÂtiment de Biologie, Université de Lausanne, Lausanne, Switzerland2

Received 8 May 2003/ Accepted 15 July 2003

High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing ~2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the periplasmic space was compressed during cryosectioning, a finding which provided supporting evidence that this space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the space between them (although still part of the periplasmic space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the periplasmic space. It is possible that the protoplast has some capacity to float freely within the periplasm.

* Corresponding author. Mailing address: Department of Microbiology, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 824-4120, ext. 53366. Fax: (519) 837-1802. E-mail: tjb{at}uoguelph.ca.

Journal of Bacteriology, October 2003, p. 6112-6118, Vol. 185, No. 20
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.20.6112-6118.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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