Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy

Fixation ability of five common fixation solutions, including 2.5% glutaraldehyde, 10% formalin, 4% paraformaldehyde, methanol/acetone (1:1), and ethanol/acetic acid (3:1) were evaluated by using atomic force microscopy in the present study. Three model bacteria, i.e., Escherichia coli, Pseudomonas putida, and Bacillus subtilis were applied to observe the above fixation methods for the morphology preservation of bacterial cells and surface ultrastructures. All the fixation methods could effectively preserve cell morphology. However, for preserving bacterial surface ultrastructures, the methods applying aldehyde fixations performed much better than those using alcohols, since the alcohols could detach the surface filaments (i.e., flagella and pili) significantly. Based on the quantitative and qualitative assessments, the 2.5% glutaraldehyde was proposed as a promising fixation solution both for observing morphology of both bacterial cell and surface ultrastructures, while the methonal/acetone mixture was the worst fixation solution which may obtain unreliable results

Multidimensional Atomic Force Microscopy: A Versatile Novel Technology for Nanopharmacology Research

Nanotechnology is giving us a glimpse into a nascent field of nanopharmacology that deals with pharmacological phenomena at molecular scale. This review presents our perspective on the use of scanning probe microscopy techniques with special emphasis to multidimensional atomic force microscopy (m-AFM) to explore this new field with a particular emphasis to define targets, design therapeutics, and track outcomes of molecular-scale pharmacological interactions. The approach will be to first discuss operating principles of m-AFM and provide representative examples of studies to understand human health and disease at the molecular level and then to address different strategies in defining target macromolecules, screening potential drug candidates, developing and characterizing of drug delivery systems, and monitoring target–drug interactions. Finally, we will discuss some future directions including AFM tip-based parallel sensors integrated with other high-throughput technologies which could be a powerful platform for drug discovery

Extracellular matrix formation enhances the ability of streptococcus pneumoniae to cause invasive disease

Extent: 17p.During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100- fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.Claudia Trappetti, Abiodun D. Ogunniyi, Marco R. Oggioni and James C. Pato