BackgroundViability staining with SYTO9 and propidium iodide (PI) is a frequently used tool in microbiological studies. However, data generated by such routinely used method are often not critically evaluated for their accuracy. In this study we aim to investigate the critical aspects of this staining method using Staphylococcus aureus and Pseudomonas aeruginosa as the model microorganisms for high throughput studies in microtiter plates. SYTO9 or PI was added alone or consecutively together to cells and the fluorescence intensities were measured using microplate reader and confocal laser scanning microscope.ResultsWe found that staining of S. aureus cells with SYTO9 alone resulted in equal signal intensity for both live and dead cells, whereas staining of P. aeruginosa cells led to 18-fold stronger signal strength for dead cells than for live ones. After counterstaining with PI, the dead P. aeruginosa cells still exhibited stronger SYTO9 signal than the live cells. We also observed that SYTO9 signal showed strong bleaching effect and decreased dramatically over time. PI intensity of the culture increased linearly with the increase of dead cell numbers, however, the maximum intensities were rather weak compared to SYTO9 and background values. Thus, slight inaccuracy in measurement of PI signal could have significant effect on the outcome.ConclusionsWhen viability staining with SYTO9 and PI is performed, several factors need to be considered such as the bleaching effect of SYTO9, different binding affinity of SYTO9 to live and dead cells and background fluorescence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0376-x) contains supplementary material, which is available to authorized users.

Ultralight and highly flexible biopolymer aerogels, composed of biomimetic cellular microstructures formed from cellulose nanofibers and silver nanowires, are assembled via a convenient and facile freeze-casting method. The lamellar, honeycomb-like, and random porous scaffolds are successfully achieved by adjusting freezing approaches to modulate the relationships between microstructures and macroscopic mechanical and electromagnetic interference (EMI) shielding performances. Combining the shielding transformation arising from in situ compression and the controlled content of building units, the optimized lamellar porous biopolymer aerogels can show a very high EMI shielding effectiveness (SE), which exceeds 70 or 40 dB in the Xband while the density is merely 6.2 or 1.7 mg/cm 3 , respectively. The corresponding normalized surface specific SE (defined as the SE divided by the material density and thickness) is up to 178235 dB•cm 2 /g, far surpassing that of the so-far reported shielding materials. Antibacterial properties and hydrophobicity are also demonstrated extending the versatility and application potential of the biopolymer hybrid aerogels.

BackgroundDue to the increased emergence of antimicrobial resistance, alternatives to minimize the usage of antibiotics become attractive solutions. Biophysical manipulation of material surface topography to prevent bacterial adhesion is one promising approach. To this end, it is essential to understand the relationship between surface topographical features and bactericidal properties in order to develop antibacterial surfaces.ResultsIn this work a systematic study of topographical effects on bactericidal activity of nanostructured surfaces is presented. Nanostructured Ormostamp polymer surfaces are fabricated by nano-replication technology using nanoporous templates resulting in 80-nm diameter nanopillars. Six Ormostamp surfaces with nanopillar arrays of various nanopillar densities and heights are obtained by modifying the nanoporous template. The surface roughness ranges from 3.1 to 39.1 nm for the different pillar area parameters. A Gram-positive bacterium, Staphylococcus aureus, is used as the model bacterial strain. An average pillar density at ~ 40 pillars μm−2 with surface roughness of 39.1 nm possesses the highest bactericidal efficiency being close to 100% compared with 20% of the flat control samples. High density structures at ~ 70 pillars μm−2 and low density structures at < 20 pillars μm−2 with surface roughness smaller than 20 nm reduce the bactericidal efficiency to almost the level of the control samples.ConclusionThe results obtained here suggests that the topographical effects including pillar density and pillar height inhomogeneity may have significant impacts on adhering pattern and stretching degree of bacterial cell membrane. A biophysical model is prepared to interpret the morphological changes of bacteria on these nanostructures.Electronic supplementary materialThe online version of this article (10.1186/s12951-018-0347-0) contains supplementary material, which is available to authorized users.

Various methods have been reported to quantify total biofilm or different components of biofilm; however, these methods are often confusedly used, leading to discrepancies and misleading results. In this study, different methods for quantification of biofilm, including those for total biomass, total amount of bacterial cells, viable cell number, and amount of extracellular polymeric substances, were systematically compared in microtiter plates. To evaluate which method is suitable for assessment of biofilm removal and for bacterial killing, biofilm samples were treated with various cleaners possessing removing and/or killing capacities. It was found that most of the methods tested in this study in general exhibited high reproducibility and repeatability. Crystal Violet staining was a simple but reliable method for total biomass quantification. Total bacteria cell numbers could be reliably quantified by the fluorescent DNA-binding dye Acridine Orange. Viable cells could be quantified by either an ATP-based assay or a proliferation assay. Both of these viability methods showed a broad detection range and led to precise measurement. For quantification of proteins in the biofilm, staining with fluorescein isothiocyanate was most suitable. Furthermore, it was revealed that a combination of different methods is required to determine if a cleaner kills or removes biofilm.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7396-9) contains supplementary material, which is available to authorized users.

We present here a technological platform for engineering Au nanotopographies by templated electrodeposition on antibacterial surfaces. Three different types of nanostructures were fabricated: nanopillars, nanorings and nanonuggets. The nanopillars are the basic structures and are 50 nm in diameter and 100 nm in height. Particular arrangement of the nanopillars in various geometries formed nanorings and nanonuggets. Flat surfaces, rough substrate surfaces, and various nanostructured surfaces were compared for their abilities to attach and kill bacterial cells. Methicillin-resistant Staphylococcus aureus, a Gram-positive bacterial strain responsible for many infections in health care system, was used as the model bacterial strain. It was found that all the Au nanostructures, regardless their shapes, exhibited similar excellent antibacterial properties. A comparison of live cells attached to nanotopographic surfaces showed that the number of live S. aureus cells was <1% of that from flat and rough reference surfaces. Our micro/nanofabrication process is a scalable approach based on cost-efficient self-organization and provides potential for further developing functional surfaces to study the behavior of microbes on nanoscale topographies.