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Evaporation-induced stimulation of bacterial osmoregulation for electrical assessment of cell viability.

ABSTRACT: Bacteria cells use osmoregulatory proteins as emergency valves to respond to changes in the osmotic pressure of their external environment. The existence of these emergency valves has been known since the 1960s, but they have never been used as the basis of a viability assay to tell dead bacteria cells apart from live ones. In this paper, we show that osmoregulation provides a much faster, label-free assessment of cell viability compared with traditional approaches that rely on cell multiplication (growth) to reach a detectable threshold. The cells are confined in an evaporating droplet that serves as a dynamic microenvironment. Evaporation-induced increase in ionic concentration is reflected in a proportional increase of the droplet's osmotic pressure, which in turn, stimulates the osmoregulatory response from the cells. By monitoring the time-varying electrical conductance of evaporating droplets, bacterial cells are identified within a few minutes compared with several hours in growth-based methods. To show the versatility of the proposed method, we show detection of WT and genetically modified nonhalotolerant cells (Salmonella typhimurium) and dead vs. live differentiation of nonhalotolerant (such as Escherichia coli DH5α) and halotolerant cells (such as Staphylococcus epidermidis). Unlike the growth-based techniques, the assay time of the proposed method is independent of cell concentration or the bacteria type. The proposed label-free approach paves the road toward realization of a new class of real time, array-formatted electrical sensors compatible with droplet microfluidics for laboratory on a chip applications.

SUBMITTER: Ebrahimi A

PROVIDER: S-EPMC4932974 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Evaporation-induced stimulation of bacterial osmoregulation for electrical assessment of cell viability.

Ebrahimi Aida A Alam Muhammad Ashraful MA

Proceedings of the National Academy of Sciences of the United States of America 20160614 26

Bacteria cells use osmoregulatory proteins as emergency valves to respond to changes in the osmotic pressure of their external environment. The existence of these emergency valves has been known since the 1960s, but they have never been used as the basis of a viability assay to tell dead bacteria cells apart from live ones. In this paper, we show that osmoregulation provides a much faster, label-free assessment of cell viability compared with traditional approaches that rely on cell multiplicati ...[more]

PMID: 27303045

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Project description:IntroductionAccurately assessing biofilm viability is essential for evaluating both biofilm formation and the efficacy of antibacterial treatments. Traditional SYTO9 and propidium iodide (PI) live/dead staining in biofilm viability assays often ace challenges due to non-specific staining, limiting precise differentiation between live and dead cells. To address this limitation, we investigated an alternative staining method employing calcein acetoxymethyl (CAM) to detect viable cells based on esterase activity, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) to assess the remaining biofilm population.MethodsBiofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecium were matured and exposed to varying concentrations of antibiotics or sterile medium. Biofilm viability was assessed using CAM/TMA-DPH or SYTO9/PIstaining, followed by analysis with confocal laser scanning microscopy (CLSM) and ImageJ-based biofilm surface coverage quantification. Viability findings were compared with colony-forming units (CFU/mL), a standard microbial viability measure.ResultsCAM/TMA-DPH staining demonstrated strong positive correlations with CFU counts across all bacterial species (r = 0.59 - 0.91), accurately reflecting biofilm vitality. In contrast, SYTO9/PI staining consistently underestimated the viability of untreated biofilms, particularly in Klebsiella pneumoniae, where a negative correlation with CFU/mL was observed (r = -0.04). Positive correlations for SYTO9/PI staining were noted in other species (r = 0.65 - 0.79). These findings underscore the limitations of membrane integrity-based staining methods and highlight the advantages of metabolic-based probes like CAM/TMA-DPH.DiscussionOur findings suggest that CAM/TMA-DPH staining provides a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms, highlighting the advantages of metabolic-based probes over traditional membrane integrity assays. The consistency of CAM/TMA-DPH staining across different bacterial species underscores its potential to advance studies on biofilm and contribute to the development of more effective anti-biofilm treatments, which is essential for clinical management of biofilm-associated infections.

Project description:Peripheral nerve injuries often lead to incomplete recovery and contribute to significant disability to approximately 360,000 people in the USA each year. Stem cell therapy holds significant promise for peripheral nerve regeneration, but maintenance of stem cell viability and differentiation potential in vivo are still major obstacles for translation. Using a made-in-house 96-well vertical electrical stimulation (ES) platform, we investigated the effects of different stimulating pulse frequency, duration and field direction on human neural crest stem cell (NCSC) differentiation. We observed dendritic morphology with enhanced neuronal differentiation for NCSCs cultured on cathodes subject to 20 Hz, 100μs pulse at a potential gradient of 200 mV/mm. We further evaluated the effect of a novel cell-based therapy featuring optimized pulsatile ES of NCSCs for in vivo transplantation following peripheral nerve regeneration. 15 mm critical-sized sciatic nerve injuries were generated with subsequent surgical repair in sixty athymic nude rats. Injured animals were randomly assigned into five groups (N = 12 per group): blank control, ES, NCSC, NCSC + ES, and autologous nerve graft. The optimized ES was applied immediately after surgical repair for 1 h in ES and NCSC + ES groups. Recovery was assessed by behavioral (CatWalk gait analysis), wet muscle-mass, histomorphometric, and immunohistochemical analyses at either 6 or 12 weeks after surgery (N = 6 per group). Gastrocnemius muscle wet mass measurements in ES + NCSC group were comparable to autologous nerve transplantation and significantly higher than other groups (p < 0.05). Quantitative histomorphometric analysis and catwalk gait analysis showed similar improvements by ES on NCSCs (p < 0.05). A higher number of viable NCSCs was shown via immunochemical analysis, with higher Schwann cell (SC) differentiation in the NCSC + ES group compared to the NCSC group (p < 0.05). Overall, ES on NCSC transplantation significantly enhanced nerve regeneration after injury and repair, and was comparable to autograft treatment. Thus, ES can be a potent alternative to biochemical and physical cues for modulating stem cell survival and differentiation. This novel cell-based intervention presents an effective and safe approach for improved outcomes after peripheral nerve repair.

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Evaporation-induced stimulation of bacterial osmoregulation for electrical assessment of cell viability.

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Evaporation-induced stimulation of bacterial osmoregulation for electrical assessment of cell viability.

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