Staphylococcus aureus is routinely recovered from air and surface samples taken aboard the International Space Station (ISS) and poses a health threat to crew. As bacteria respond to the low shear forces engendered by continuous rotation conditions in a Rotating Wall Vessel (RWV) and the reduced gravitational field of near-Earth flight by altering gene expression, we examined the effect of low-shear RWV growth on protein secretion and gene expression by three S. aureus isolates. When cultured under 1 g, the total amount of protein secreted by these strains varied up to fourfold; under continuous rotation conditions, protein secretion by all three strains was significantly reduced. Concentrations of individual proteins were differentially reduced and no evidence was found for increased lysis. These data suggest that growth under continuous rotation conditions reduces synthesis or secretion of proteins. A limited number of changes in gene expression under continuous rotation conditions were noted: in all isolates vraX, a gene encoding a polypeptide associated with cell wall stress, was down-regulated. A vraX deletion mutant of S. aureus SH1000 was constructed: no differences were found between SH1000 and ΔvraX with respect to colony phenotype, viability, protein export, antibiotic susceptibility, vancomycin kill kinetics, susceptibility to cold or heat and gene modulation. An ab initio protein–ligand docking simulation suggests a major binding site for β-lactam drugs such as imipenem. If such changes to the bacterial phenotype occur during spaceflight, they will compromise the capacity of staphylococci to cause systemic infection and to circumvent antibacterial chemotherapy.

The three-dimensional (3D) keratin filament network of pancreatic carcinoma cells was investigated with different electron microscopical approaches. Semithin sections of high-pressure frozen and freeze substituted cells were analyzed with scanning transmission electron microscope (STEM) tomography. Preservation of subcellular structures was excellent, and keratin filaments could be observed; however, it was impossible to three-dimensionally track the individual filaments. To obtain a better signal-to-noise ratio in transmission mode, we observed ultrathin sections of high-pressure frozen and freeze substituted samples with low-voltage (30 kV) STEM. Contrast was improved compared to 300 kV, and individual filaments could be observed. The filament network of samples prepared by detergent extraction was imaged by high-resolution scanning electron microscopy (SEM) with very good signal-to-noise ratio using the secondary electron signal and the 3D structure could be elucidated by SEM tomography. In freeze-dried samples it was possible to discern between keratin filaments and actin filaments because the helical arrangement of actin subunits in the F-actin could be resolved. When comparing the network structures of the differently prepared samples, we found no obvious differences in filament length and branching, indicating that the intermediate filament network is less susceptible to preparation artifacts than the actin network.

The impact of rehydration on a multicellular organism was studied in lettuce (Lactuca sativa L.) embryos, using cryo-scanning electron microscopy (cryo-SEM). Naked embryos were sensitive to imbibitional stress, whereas embryos with an intact, thick-walled endosperm were not. Imbibitional injury to naked embryos was mainly confined to the outer 2–3 cell layers of the axis. These cells failed to swell and appeared poorly hydrated. Deeper layers were not affected even after extended periods of cold rehydration. The proportion of damaged cells (6–7% of total) roughly corresponded with the additional K+ that gradually leached from the embryos. Damaged embryos were able to survive the loss of their surface layers and form adventitious roots. The swelling of inner tissues caused the dead surface layers to rupture into patches. Plasma membranes in dried embryos showed normal bilayer structure with a homogeneous distribution of intra-membrane particles (IMPs), also after non-injurious rehydration. Imbibitionally damaged plasma membranes showed many irregularities, such as globular insertions, that probably resulted from malfusions in the ruptured membrane, but the IMPs were still randomly distributed.

Recent studies have provided evolutionary explanations for much of the variation in mortality among human infectious diseases. One gap in this knowledge concerns respiratory tract pathogens transmitted from person to person by direct contact or through environmental contamination. The sit-and-wait hypothesis predicts that virulence should be positively correlated with durability in the external environment because high durability reduces the dependence of transmission on host mobility. Reviewing the epidemiological and medical literature, we confirm this prediction for respiratory tract pathogens of humans. Our results clearly distinguish a high-virulence high-survival group of variola (smallpox) virus, Mycobacterium tuberculosis, Corynebacterium diphtheriae, Bordetella pertussis, Streptococcus pneumoniae, and influenza virus (where all pathogens have a mean percent mortality [ges ]0.01% and mean survival time >10 days) from a low-virulence low-survival group containing ten other pathogens. The correlation between virulence and durability explains three to four times of magnitude of difference in mean percent mortality and mean survival time, using both across-species and phylogenetically controlled analyses. Our findings bear on several areas of active research and public health policy: (1) many pathogens used in the biological control of insects are potential sit-and-wait pathogens as they combine three attributes that are advantageous for pest control: high virulence, long durability after application, and host specificity; (2) emerging pathogens such as the ‘hospital superbug’ methicillin-resistant Staphylococcus aureus (MRSA) and potential bioweapons pathogens such as smallpox virus and anthrax that are particularly dangerous can be discerned by quantifying their durability; (3) hospital settings and the AIDS pandemic may provide footholds for emerging sit-and-wait pathogens; and (4) studies on food-borne and insect pathogens point to future research considering the potential evolutionary trade-offs and genetic linkages between virulence and durability.

High-pressure frozen rat pancreas tissue samples were cryo-fractured and cryo-sectioned with a diamond knife in the microtome of a freeze-etching device. The bulk fracture faces and block faces were investigated in the frozen-hydrated state by use of a cryo-stage in an in-lens SEM. With this combination, relevant biological structures can be investigated with a few nanometers resolution in a near lifelike state, preventing the artifacts of conventional fixation techniques. Compared to TEM replica techniques, the presented method is more versatile since no replica cleaning is necessary. Additional structures can be made visible by controlled sublimation of ice, leading to a better understanding of the three-dimensional organization of organelles, such as the endoplasmic reticulum.

The exposure to discharging nematocysts of the eolid nudibranch Cratena peregrina feeding on polyps of the hydroid Eudendrium racemosum was examined. The smaller size-class of microbasic eurytele mastigophor nematocysts is located on the tentacles of the hydroid polyps, while the larger size-class of holotrichous isorhiza nematocysts is located on long cnidophores and basal rings of some polyps. Discharging nematocysts of the tentacles do not appear to have damaging effects on the dorsal appendages of the snail, the cerata. In contrast, contact of a cnidophore with a ceras elicits massive discharge of nematocysts, which results in adherence, membranolysis and local destruction of the epidermis within seconds. The differential locations and effects indicate that the small tentacle nematocysts serve for capturing prey, while the large cnidophore nematocysts serve for the hydroid's defence. Both small and large, discharged and intact nematocysts were found in the pharynx and stomach of the snail after feeding. Thus, the alimentary tract appears to be efficiently protected against discharging nematocysts. Small and large intact nematocysts, which were capable of discharge, were also found in the cnidosacs.

Imaging of fast frozen samples is the most direct approach for electron microscopy of biological specimen in a defined physiological state. It prevents chemical fixation and drying artifacts. High pressure freezing allows for ice-crystal-free cryo-fixation of tissue pieces up to a thickness of 200 urn and a diameter of 2 mm without prefixation. Such a frozen disc, however, is not directly amenable to electron microscopic observation: The structures of interest have to be made amenable to the electron beam, and the structures of interest must produce enough contrast to be recognized in the electron microscope. This can be achieved by freeze fracturing, cryo-sectioning or freeze substitution.

The figures show high pressure frozen bakers yeast saccharomyces cerevisiae in the cryo-SEM (Figures 1 and 2) and after freeze substitution in the TEM (Figure 3). For high pressure freezing either a Bal-Tec HPM 010 (Princ. of Liechtenstein; Figures 1 and 2), or a Wohlwend HPF (Wohlwend GmbH, Sennwald, Switzerland; Figure 3) were used.

Joy and Pawley (1992) defined the limitations of spatial resolution in the SEM as follows. “The spatial resolution of the scanning electron microscope is limited by at least three factors: the diameter of the electron probe, the size and shape of the beam/specimen interaction volume with the solid for the mode of imaging employed and the Poisson statistics of the detected signal.” The interaction volume of the primary beam in the sample increases with increasing accelerating voltage (Vo), because electrons with higher energy penetrate deeper into the sample. The interaction volume affects the spatial resolution, because the electrons that finally form the image do not only originate from the impact point of the primary beam, but from the much larger interaction volume. It is not simple to understand how this affects spatial resolution, since resolution is also heavily influenced by the properties of the sample surface (the surface coating) and it is the issue of ongoing debates.

High-pressure facilitates freezing of biological specimens: pressurizing to 2000 bar before cooling retards growth and reduces size of ice crystals in the specimen. Although the lung is 80% air and is generally difficult to freeze, we report here good cellular ultrastructure in high-pressure frozen lung when: 1) the lung is allowed to collapse (empty of air) prior to sampling, or 2) air-filled lung is allowed to compress during pressurization in the high-pressure freezer prior to freezing or 3) airspaces are filled with relatively incompressible fluorocarbon liquid.

Small Balb mice (28-30g) were anaesthetized and ventilated via endotracheal tube. The chest was opened and a 2mm diameter disk of lung at the thin anterior margin of the right lung was clamped between two aluminum tophats (200μm wells), sealing in air. In other experiments, the lung was allowed to collapse to 0 cm water transpulmonary pressure prior to clamping. In the third series of experiments, air was gradually replaced with fluorocarbon liquid (FC5312, 3M, Minneapolis MN) and the lung inflated with liquid to functional residual capacity. The top hat assembly containing the sealed lung was transferred to the high-pressure freezer (HPM 010, Bal-Tec, Balzers, Lichtenstein) where they were pressurized and frozen.

Sample preparation techniques for electron-microscopy (EM) dictate our perception of the microworld: any structural information which is lost or distorted during preparation can not be regenerated later and might lead to wrong interpretation of the observed micrograph.

Cryofixation based procedures for specimen preparation can avoid most of the structural alterations associated with standard techniques based on chemical fixation. The ultrastructure can be represented in a near “native state” thanks to the high time resolution for dynamic cellular events (1).

High pressure freezing (2) permits to cryoimmobilize biological samples up to approx. 200μm thick, in contrast to rapid freezing procedures at ambient pressure that are useful to cryoimmobilize samples up to 10-20 μm thick. The actual samplethickness that can be adequately frozen (=without visible damage due to ice crystal formation in freeze-substituted or freeze-fractured specimens) depends on the concentration of naturally occuring substances that exhibit cryoprotective activities.