The application of STEM and in situ controlled dehydration to bacterial systems using ESEM

Transmission imaging with an environmental scanning electron microscope (ESEM) (Wet STEM) is a recent development in the field of electron microscopy, combining the simple preparation inherent to ESEM work with an alternate form of contrast available through a STEM detector. Because the technique is relatively new, there is little information available on how best to apply this technique and which samples it is best suited for. This work is a description of the sample preparation and microscopy employed by the authors for imaging bacteria with Wet STEM (scanning transmission electron microscopy). Three different bacterial samples will be presented in this study: first, used as a model system, is Escherichia coli for which the contrast mechanisms of STEM are demonstrated along with the visual effects of a dehydration-induced collapse. This collapse, although clearly in some sense artifactual, is thought to lead to structurally meaningful morphological information. Second, Wet STEM is applied to two distinct bacterial systems to demonstrate the novel types of information accessible by this approach: the plastic-producing Cupriavidus necator along with wild-type and δmreC knockout mutants of Salmonella enterica serovar Typhimurium. Cupriavidus necator is shown to exhibit clear internal differences between bacteria with and without plastic granules, while the δmreC mutant of S. Typhimurium has an internal morphology distinct from that of the wild type. © 2012 Wiley Periodicals, Inc.

Attenuated Salmonella Typhimurium lacking the pathogenicity island-2 type 3 secretion system grow to high bacterial numbers inside phagocytes in mice.

Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics.

Evaluating the impact of V2G services on the degradation of batteries in PHEV and EV

Many researchers and industry observers claim that electric vehicles (EV) and plug-in hybrid electric vehicles (PHEV) could provide vehicle-to-grid (V2G) bulk energy and ancillary services to an electricity network. This work quantified the impact on various battery characteristics whilst providing such services. The sensitivity of the impact of V2G services on battery degradation was assessed for EV and PHEV for different battery capacities, charging regimes, and battery depth of discharge. Battery degradation was found to be most dependent on energy throughput for both the EV and PHEV powertrains, but was most sensitive to charging regime (for EVs) and battery capacity (for PHEVs). When providing ancillary services, battery degradation in both powertrains was most sensitive to individual vehicle battery depth of discharge. Degradation arising from both bulk energy and ancillary services could be minimised by reducing the battery capacity of the vehicle, restricting the number of hours connected and reducing the depth of discharge of each vehicle for ancillary services. Regardless, best case minimum impacts of providing V2G services are severe such as to require multiple battery pack replacements over the vehicle lifetime. © 2013 Elsevier Ltd.