Chapter 11 - Cryo-Electron Microscopy of Extremely Halophilic Microbes

Publication date: 2018Source: Methods in Microbiology, Volume 45Author(s): Harold Engelhardt, Daniel BollschweilerAbstractHalophilic and extremely halophilic microorganisms in particular have been a challenge for conventional electron microscopy (EM) as well as for state-of-the-art cryo-EM and cryo-electron tomography (cryo-ET). Conventional preparation techniques such as negative staining of isolated macromolecular complexes and plastic embedding of whole cells for ultrathin sectioning suffer from the problem of eliminating most of the salt without destroying the biological samples. Cryo-EM on the other hand, which is intended for dealing with close-to-life conditions of halophiles, is confronted with samples containing several molar NaCl in media in the extreme case, resulting in low contrast and high noise images. The latter has prevented cryo-ET of extreme halophiles, excluding a large group of microbes from structural investigations in situ. The survey of EM applications with salt-containing specimens and the systematic evaluation of cryopreparations and cryo-EM of Halobacterium salinarum in 3 M NaCl showed that extreme halophiles can be studied under close-to-life conditions. This review of methods compiles useful strategies for corresponding approaches.
Source: Methods in Microbiology - Category: Microbiology Source Type: research