BackgroundThe Double-Layer Agar (DLA) technique is extensively used in phage research to enumerate and identify phages and to isolate mutants and new phages. Many phages form large and well-defined plaques that are easily observed so that they can be enumerated when plated by the DLA technique. However, some give rise to small and turbid plaques that are very difficult to detect and count. To overcome these problems, some authors have suggested the use of dyes to improve the contrast between the plaques and the turbid host lawns. It has been reported that some antibiotics stimulate bacteria to produce phages, resulting in an increase in final titer. Thus, antibiotics might contribute to increasing plaque size in solid media.ResultsAntibiotics with different mechanisms of action were tested for their ability to enhance plaque morphology without suppressing phage development. Some antibiotics increased the phage plaque surface by up to 50-fold.ConclusionThis work presents a modification of the DLA technique that can be used routinely in the laboratory, leading to a more accurate enumeration of phages that would be difficult or even impossible otherwise.

Whey valorization concerns have led to recent interest on the production of whey beverage simulating kefir. In this study, the structure and microbiota of Brazilian kefir grains and beverages obtained from milk and whole/deproteinised whey was characterized using microscopy and molecular techniques. The aim was to evaluate its stability and possible shift of probiotic bacteria to the beverages. Fluorescence staining in combination with Confocal Laser Scanning Microscopy showed distribution of yeasts in macro-clusters among the grain's matrix essentially composed of polysaccharides (kefiran) and bacteria. Denaturing gradient gel electrophoresis displayed communities included yeast affiliated to Kluyveromyces marxianus, Saccharomyces cerevisiae, Kazachatania unispora, bacteria affiliated to Lactobacillus kefiranofaciens subsp. Kefirgranum, Lactobacillus kefiranofaciens subsp. Kefiranofaciens and an uncultured bacterium also related to the genus Lactobacillus. A steady structure and dominant microbiota, including probiotic bacteria, was detected in the analyzed kefir beverages and grains. This robustness is determinant for future implementation of whey-based kefir beverages.

Bacteria are simple organisms with a remarkable capacity for survival by adapting to different environments, which is a result of their long evolutionary history. Taking into consideration these adapting mechanisms, this work now investigates the effect of electrically active microenvironments on bacteria and on how this stimulation may trigger bacteria growth inhibition or proliferation. Electrical microenvironments are generated via stimulation of a piezoelectric polymer with a mechanical cue, thus developing an electrical response and a variation on the surface charge of the polymeric material. Specifically, Grampositive Staphylococcus epidermidis and Gram-negative Escherichia coli were grown overnight under static and dynamic conditions on piezoelectric poly(vinylidene) fluoride (PVDF) films to further study bacteria behavior under: (i) the effect of the material surface charge in static conditions, (ii) the mechanical effect, and (iii) the piezoelectric effect, the last two performed under dynamic conditions. Bacteria viability in planktonic and biofilm forms was measured, and the microorganism morphology was characterized. Whereas E. coli responds little to any of the stimuli application, S. epidermidis growth can be regulated through the material surface charge and by the applied frequency. Positively charged PVDF induces bacterial growth inhibition in planktonic and adhered cells in static conditions, whereas antifouling properties are obtained when a mechanical or piezoelectric effect at 4 Hz stimuli is applied. By increasing the stimuli to 40 Hz, however, the adhesion of bacteria is promoted. In conclusion, the behavior of certain bacteria species is tailored through the application of piezoelectric materials, which provide sufficient mechanoelectrical stimuli for growth or inhibition of bacteria, allowing for the design of suitable anti-and promicrobial strategies. Such strategies are only found in studies related to mammalian cells, whereas in bacterial cells this type of stimuli are still unknown. Thus, this work provides one of the first insights on the effect of piezoelectric stimuli on bacterial cells.