Cryopreservation of swine colostrum-derived cells.

Mesenchymal stromal cells (MSCs) have been demonstrated to possess anti-inflammatory and antimicrobial properties and are of interest in biotechnologies that will require cryopreservation. Recently, MSC-like cells were isolated from colostrum and milk. We used an interrupted slow freezing procedure to examine cryoinjury incurred during slow cooling and rapid cooling of MSC-like cells from swine colostrum. Cells were loaded with either dimethyl sulfoxide (Me2SO) or glycerol, cooled to a nucleation temperature, ice-nucleated, and further cooled at 1 °C/min. At several temperatures along the cooling path, cells were either thawed directly, or plunged into liquid nitrogen for storage and later thawed. The pattern of direct-thaw and plunge-thaw responses was used to guide optimization of cryopreservation protocol parameters. We found that both 5% Me2SO (0.65 M, loaded for 15 min on ice) or 5% glycerol (0.55 M, loaded for 1 h at room temperature) yielded cells with high post-thaw membrane integrity when cells were cooled to at least -30 °C before being plunged into, and stored in, liquid nitrogen. Cells cultured post-thaw exhibited osteogenic differentiation similar to fresh unfrozen control. Fresh and cryopreserved MSC-like cells demonstrated antimicrobial activity against S. aureus. Also, the antimicrobial activity of cell-conditioned media was higher when both fresh and cryopreserved MSC-like cells were pre-exposed to S. aureus. Thus, we were able to demonstrate cryopreservation of colostrum-derived MSC-like cells using Me2SO or glycerol, and show that both cryoprotectants yield highly viable cells with osteogenic potential, but that cells cryopreserved with glycerol retain higher antimicrobial activity post-thaw.

Atomic force microscopy: a nanoscopic view of microbial cell surfaces.

The atomic force microscope (AFM) is a powerful instrument for microbiological investigation. It has evolved from an imaging tool used to investigate microbial surfaces at high resolution in their physiological environment into a lab-on-a-tip device, which allows more quantitative analysis of biological samples (from molecules to cells) in aqueous liquids. Atomic force microscopy provides information about the nanoscale architecture of microbes and about the localization and interactions of their individual constituents. Microbial interactions play essential roles in biology, medicine, ecology, biotechnology, food science and contribute to phenomena as varied as bacterial infections, biofilm formation, and bacterial adhesion to surfaces. In this review, we focus on recent developments offered by the rapid advances in AFM imaging and force spectroscopy with emphasizes on microbial research.

Selective sorting of cargo proteins into bacterial membrane vesicles.

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.

Application of atomic force microscopy in bacterial research.

The atomic force microscope (AFM) has evolved from an imaging device into a multifunctional and powerful toolkit for probing the nanostructures and surface components on the exterior of bacterial cells. Currently, the area of application spans a broad range of interesting fields from materials sciences, in which AFM has been used to deposit patterns of thiol-functionalized molecules onto gold substrates, to biological sciences, in which AFM has been employed to study the undesirable bacterial adhesion to implants and catheters or the essential bacterial adhesion to contaminated soil or aquifers. The unique attribute of AFM is the ability to image bacterial surface features, to measure interaction forces of functionalized probes with these features, and to manipulate these features, for example, by measuring elongation forces under physiological conditions and at high lateral resolution (<1 A). The first imaging studies showed the morphology of various biomolecules followed by rapid progress in visualizing whole bacterial cells. The AFM technique gradually developed into a lab-on-a-tip allowing more quantitative analysis of bacterial samples in aqueous liquids and non-contact modes. Recently, force spectroscopy modes, such as chemical force microscopy, single-cell force spectroscopy, and single-molecule force spectroscopy, have been used to map the spatial arrangement of chemical groups and electrical charges on bacterial surfaces, to measure cell-cell interactions, and to stretch biomolecules. In this review, we present the fascinating options offered by the rapid advances in AFM with emphasizes on bacterial research and provide a background for the exciting research articles to follow.

Analysis of force interactions between AFM tips and hydrophobic bacteria using DLVO theory.

Microbial adhesion to surfaces and interfaces is strongly influenced by their structure and physicochemical properties. We used atomic force microscopy (AFM) to measure the forces between chemically functionalized AFM tips and two bacterial species exhibiting different cell surface hydrophobicities, measured as the oil/water contact angle (theta): Acinetobacter venetianus RAG-1 (theta = 56.4 degrees ) and Rhodococcus erythropolis 20S-E1-c (theta = 152.9 degrees ). The forces were measured as the AFM tips, coated with either hydrophobic (octadecane) or hydrophilic (undecanol) groups, approached the bacterial cells in aqueous buffer. The experimental force curves between the two microbial cells and functionalized AFM probes were not successfully described by the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloid stability. To reconcile the discrepancy between theory and experiments, two types of extended DLVO models were proposed. The first modification considers an additional acid-base component that accounts for attractive hydrophobic interactions and repulsive hydration effects. The second model considers an additional exponentially decaying steric interaction between polymeric brushes in addition to the acid-base interactions. These extended DLVO predictions agreed well with AFM experimental data for both A. venetianus RAG-1, whose surface consists of an exopolymeric capsule and pili, and R. erythropolis 20S-E1-c, whose surface is covered by an exopolymeric capsule. The extended models for the bacteria-AFM tip force-distance curves were consistent with the effects of steric interactions.

Atomic force microscopy measurement of heterogeneity in bacterial surface hydrophobicity.

The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.

Stabilization of oil-water emulsions by hydrophobic bacteria.

Formation of oil-water emulsions during bacterial growth on hydrocarbons is often attributed to biosurfactants. Here we report the ability of certain intact bacterial cells to stabilize oil-in-water and water-in-oil emulsions without changing the interfacial tension, by inhibition of droplet coalescence as observed in emulsion stabilization by solid particles like silica.