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Dissolved organic matter in open-canopy streams is mostly derived from autochthonous sources, but the role of autochthonous DOM to sustain heterotrophic communities has been poorly studied. We compared respiration rates of epiphytic and epipelic biofilms (measured by the Resazurin—Resorufin tracer system) when processing autochthonous and allocthonous DOM sources with different chemical and optical properties. We performed laboratory experiments to determine aerobic respiration rates of epipelic and epiphytic communities in different leachates from autochthonous (macroalgae and emergent and submerged macrophytes) and allochthonous (riparian grasses) DOM sources from a Pampean stream. We also determined dissolved organic carbon and nutrient contents and DOM optical properties of each leachate. We found that epipelon was more heterotrophic and showed a lower respiratory activity than epiphyton, regardless of the type of leachate. Epiphytic respiratory activity was positively associated with DOM of microbial origin with a lower degree of humification (i.e., autochthonous microbial-algal derived materials), while epipelic respiratory activity was related to autochthonous and allochthonous humic-like materials, with higher aromaticity, derived from grasses and macrophytes. This indicates that both autochthonous and allochthonous sources can sustain microbial respiratory activity in the stream, and highlights the role of primary producers in the carbon cycle of open-canopy streams. [ABSTRACT FROM AUTHOR]

Background in vitro susceptibility testing for the non-sporulating fungus Madurella mycetomatis is performed with a hyphal suspension as starting inoculum and a viability dye for endpoint reading. Here we compared the performance of four different viability dyes for their use in in vitro susceptibility testing of M. mycetomatis. Methods To compare the reproducibility and the agreement between the viability dyes 2,3-bis-(2-methoxy-4-nitro-5-sulfphenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), resazurin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and luciferin, the in vitro susceptibilities of 14 genetically diverse M. mycetomatis isolates were determined for itraconazole and amphotericin B. The reproducibility, agreement, price and ease of use were compared. Results Each of the four dyes gave highly reproducible results with >85.7% reproducibility. Percentage agreement ranged between 78.9% and 92.9%. Resazurin was the most economical to use (0.0009 €/minimal inhibitory concentration [MIC]) and could be followed in real time. Luciferin omitted the need to transfer the supernatant to a new 96-well plate, but cost 6.07 €/MIC. Conclusion All four viability dyes were suitable to determine the in vitro susceptibility of M. mycetomatis against itraconazole and amphotericin B. Based on the high reproducibility, high percentage agreement, price and possibility to monitor in real time, resazurin was the most suited for routine in vitro susceptibility testing in the diagnostic laboratory in mycetoma-endemic countries. Because luminescence could be measured directly without the need to transfer the supernatant to a new 96-well plate, luciferin is suitable for drug-screening campaigns. Lay summary To determine the in vitro susceptibility testing in the non-sporulating fungus Madurella mycetomatis , a viability dye is needed for endpoint reading. In this study we tested the viability dyes XTT, resazurin, MTS and luciferin for their use in in vitro susceptibility testing. It appeared that they all could be used but there were differences in time to result and costs associated with them. [ABSTRACT FROM AUTHOR]

Nanomaterial-mediated plant genetic engineering holds promise for developing new crop cultivars but can be hindered by nanomaterial toxicity to protoplasts. We present a fast, high-throughput method for assessing protoplast viability using resazurin, a non-toxic dye converted to highly fluorescent resorufin during respiration. Protoplasts isolated from hypocotyl canola (Brassica napus L.) were evaluated at varying temperatures (4, 10, 20, 30 ˚C) and time intervals (1–24 h). Optimal conditions for detecting protoplast viability were identified as 20,000 cells incubated with 40 µM resazurin at room temperature for 3 h. The assay was applied to evaluate the cytotoxicity of silver nanospheres, silica nanospheres, cholesteryl-butyrate nanoemulsion, and lipid nanoparticles. The cholesteryl-butyrate nanoemulsion and lipid nanoparticles exhibited toxicity across all tested concentrations (5-500 ng/ml), except at 5 ng/ml. Silver nanospheres were toxic across all tested concentrations (5-500 ng/ml) and sizes (20–100 nm), except for the larger size (100 nm) at 5 ng/ml. Silica nanospheres showed no toxicity at 5 ng/ml across all tested sizes (12–230 nm). Our results highlight that nanoparticle size and concentration significantly impact protoplast toxicity. Overall, the results showed that the resazurin assay is a precise, rapid, and scalable tool for screening nanomaterial cytotoxicity, enabling more accurate evaluations before using nanomaterials in genetic engineering. [ABSTRACT FROM AUTHOR]

Abstract Nanomaterial-mediated plant genetic engineering holds promise for developing new crop cultivars but can be hindered by nanomaterial toxicity to protoplasts. We present a fast, high-throughput method for assessing protoplast viability using resazurin, a non-toxic dye converted to highly fluorescent resorufin during respiration. Protoplasts isolated from hypocotyl canola (Brassica napus L.) were evaluated at varying temperatures (4, 10, 20, 30 ˚C) and time intervals (1–24 h). Optimal conditions for detecting protoplast viability were identified as 20,000 cells incubated with 40 µM resazurin at room temperature for 3 h. The assay was applied to evaluate the cytotoxicity of silver nanospheres, silica nanospheres, cholesteryl-butyrate nanoemulsion, and lipid nanoparticles. The cholesteryl-butyrate nanoemulsion and lipid nanoparticles exhibited toxicity across all tested concentrations (5-500 ng/ml), except at 5 ng/ml. Silver nanospheres were toxic across all tested concentrations (5-500 ng/ml) and sizes (20–100 nm), except for the larger size (100 nm) at 5 ng/ml. Silica nanospheres showed no toxicity at 5 ng/ml across all tested sizes (12–230 nm). Our results highlight that nanoparticle size and concentration significantly impact protoplast toxicity. Overall, the results showed that the resazurin assay is a precise, rapid, and scalable tool for screening nanomaterial cytotoxicity, enabling more accurate evaluations before using nanomaterials in genetic engineering.

This study aimed to evaluate the phenolic profile, antioxidant, antibacterial, and antidiabetic properties of ethanolic and methanolic extracts of the different parts of the endemic Odontarrhena floribunda. The antioxidant activities of O. floribunda were determined with 1,1-diphenyl-1-picrylhydrazyl (DPPH) and the copper-reducing antioxidant capacity (CUPRAC) assays. The strongest antioxidant activity was recorded in methanolic root extract (IC50:1.68±0.25 μg/mL) for DPPH and ethanolic root (λ450:0.68±0.20) extract for CUPRAC. Antibacterial activities of the extracts were displayed by the Resazurin Microplate Method (REMA). All plant extracts analyzed demonstrated stronger effects on Acinetobacter baumannii (ATCC 02026) than those of the standard compound. The in vitro α-amylase and α-glucosidase inhibitory of the plant extract were assessed using the Iodine method. Except for the methanol leaf extract, all other extracts showed strong α-glucosidase inhibitory activity (0.88–2.13 mmolTE/g). These results show that this endemic O. floribunda is a potential natural antioxidant, antibacterial, and enzyme inhibitor source. [ABSTRACT FROM AUTHOR]

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z′ factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass. [ABSTRACT FROM AUTHOR]

Natural killer cell‐derived extracellular vesicles (NK‐EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK‐EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine‐based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK‐EVs against leukaemia K562 cells (suspension model) and breast cancer MDA‐MB‐231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin‐based cell viability potency assay reliably and reproducibly measured a dose‐response of NK‐EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra‐run and inter‐run variability. The assay signal showed specificity and selectivity of NK‐EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5‐hour treatment with NK‐EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA‐MB‐231 cells per test with a consistent range. Importantly, NK‐EVs' dose‐response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK‐EVs' corona (FasL, GNLY, GzmB, PFN and IFN‐γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK‐EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA‐binding reporter assay and confluence assay), our results support this highly sensitive resazurin‐based viability potency assay as a high‐throughput and quantitative method for assessing NK‐EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK‐EVs' biotherapeutics. [ABSTRACT FROM AUTHOR]

Bioburden detection is crucial for food, water, and biopharmaceutical applications as it can directly impact public health. The objective of this study is to develop and validate an assay and protocol for detecting bioburden on solid surfaces, as well as in water, with high sensitivity and accuracy in a rapid manner. Henceforth, a resazurin‐based assay optimized for detecting bioburden has been integrated with a previously developed portable multichannel fluorometer. The microbes were isolated from solid surfaces in different laboratory settings by swabbing technique, and stream water was collected for contamination analysis. Based on the results, the assay and protocol can successfully detect bioburden as low as 20 CFU/cm2 and 10 CFU/mL present in both surface and water samples, respectively. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the antibiotic resistance of five foodborne Escherichia coli clinical isolates to ciprofloxacin, gentamicin and ampicillin. Methods: Based on the theory of electron transfer during bacterial energy metabolism and the introduction of resazurin as a redox probe, an electrochemical square wave voltammetry （SWV） method based on bladed tensin-mediated rapid identification of E. coli drug resistance was established. Results: The electrochemical square wave voltammetry values of E. coli at different concentrations mediated by resazurin were determined, and there was a good linear relationship between the E. coli concentration and the oxidized peak current （I） of resazurin in the range of 1.69×102～1.69×108 CFU/mL, with the regression equation of Y=-0.156 0X-2.965 5, and R2=0.977 5; When performing the determination of E. coli drug resistance, the drug sensitivity test standard was developed by Clinical and Laboratory Standards Institute （CLSI）. The oxidized peak current （I0） of resazurin and the oxidized peak current （I-a） obtained by E. coli after directly interacting with resazurin were used as the control, and the oxidized peak current （I+a） obtained by E. coli after treating it with antibiotics and interacting with resazurin was determined. The value of bacterial activity VETA was introduced as VETA as an indicator for the identification of bacterial resistance by electrochemical square wave voltammetry, and established a rapid electrochemical drug sensitivity test method for E. coli mediated by resazurin. The established method was used to detect the resistance of five clinical isolates of foodborne E. coli to three antibiotics, and VETA=50 was used as the critical value for determining bacterial sensitivity and resistance, and the results of the drug sensitivity tests were all in accordance with the results of the paper diffusion method. The VETA=50 was used as the critical value for determining bacterial susceptibility and resistance, and all the results were consistent with the results of the paper diffusion method, and the paper diffusion method required 18 h to report the results, whereas this method required only 2 h. Conclusion: Our electrochemical square-wave voltammetry method based on the resazurin-mediated rapid discrimination of E. coli drug resistance is able to accurately and rapidly detect the antibiotics resistance of the five foodborne clinical isolates of E. coli to ciprofloxacin, gentamicin, and ampicillin.

The resazurin test is one of the most widespread approaches for studying the growth and metabolic activity of microorganisms. It is based on the colour change of the blue indicator, resazurin, to its pink reduced form, resorufin due to the reduction process catalyzed by the metabolic activity. At the same time, the quantitative characterization of the process needs to take into account the fact that one registers the results of the chemical transformation, which can differ from the underlying kinetics of the population growth. Purpose. The principal goal of this work is a sequential modelling of both coupled nonlinear growth processes aimed at obtaining the closed-form solution depending on the specificity and parameters of biological and chemical counterparts and its comparison with the curves obtained experimentally. Methods. The indicator concentration change is derived under the assumption of the logistic bacterial growth catalyzing the unidirectional chemical reaction considered and compared with the photometrically registered growth curve for a population of lactobacteria. Results. It is revealed that the biochemical growth curve will be logistic too only in the case of specially coordinated kinetic parameters and the systems’ carrying capacity. Otherwise, another functional form should be used to approximate the observable dynamics. Conclusion. Thus, the main conclusion consists of drawing attention to the importance of distinguishing between the underlying microbial and observable chemical growth curves. Their difference affects the value of the population growth rate, which is the target of such tests, and, therefore, the proper functional form should be used for the experimental data regression.

Background Colistin-resistant Acinetobacter baumannii isolates are extremely important pathogens for hospital-acquired infections. Objective To investigate the effectiveness of the resazurin microplate assay (REMA) for the rapid determination of colistin resistance. Methods Susceptibility for colistin was investigated in vitro by the broth microdilution method (BMD) and the resazurin microplate assay (REMA) on 106 carbapenem-resistant Acinetobacter baumannii isolates. Results The results of both test methods were compared, and the categorical agreement between them was found to be 100%. No minor, major, or very major discrepancy was observed between the 2 methods. Conclusions The most important advantages of REMA are that the results are obtained within 6 hours compared to the reference method, that it is easy to evaluate because it is colorimetric, and that the susceptibility result can be reported to the clinician on the same day as bacterial identification. [ABSTRACT FROM AUTHOR]

Corynebacterium pseudotuberculosis causes infection in goats, sheep, cattle, and humans. The disease imposing huge economic losses such as reducing milk, meat, and wool production and downgrading and condemnation of affected carcass to goat-rearing industry. Despite all its devastating effects, no effective antimicrobial was suggested for the treatment of this pathogen due to its highly resistant nature. Development and utilization of nanoparticles has paved the way to revive the efficacy of conventional antibiotics. Encapsulation of antibiotics within liposome as a nanoparticle may present a potential approach for successful treatment of resistant bacterial infections. The aim of this research was therefore to evaluate the antibacterial activity of free and liposome-encapsulating tylosin against Corynebacterium pseudotuberculosis. The minimum inhibitory concentration of free and liposome-encapsulated tylosin was determined by microbroth dilution and resazurin colorimetric-based methods. The antibiofilm activity of free and liposomal tylosin was assessed by crystal violet and resazurin techniques. Acridine orange/ethidium bromide staining assay was performed to assess the effect of free and liposomal tylosin on pre-formed biofilm of Corynebacterium pseudotuberculosis. The findings demonstrated minimum inhibitory concentration value of 1 µg/mL and 32 µg/mL for free and liposomal tylosin, respectively. The biofilm inhibitory data showed that free tylosin inhibited the formation of Corynebacterium pseudotuberculosis at 2 µg/mL, while this concentration was 64 µg/mL for liposomal tylosin. The antibiofilm concentration was 1024 µg/mL and 2048 µg/mL for liposome-encapsulated and free tylosin, respectively. It can be concluded that liposome as a nanocarrier can offer an excellent system of drug delivery for enhancing the efficacy of antibiotics against resistant form of Corynebacterium pseudotuberculosis. [ABSTRACT FROM AUTHOR]

Neisseria gonorrhoeae has developed resistance to every antibiotic currently approved for the treatment of gonorrhea, prompting the development of new therapies. The phenoxazine dye resazurin exhibits robust antimicrobial activity against N. gonorrhoeae in vitro but fails to limit vaginal colonization by N. gonorrhoeae in a mouse model. The lack of in vivo efficacy may be due to oxygen limitation as in vitro susceptibility assays with resazurin are conducted under atmospheric oxygen while a microaerophilic environment is present in the vagina. Here, we utilized broth microdilution assays to determine the susceptibility of N. gonorrhoeae to resazurin under low and atmospheric oxygen conditions. The minimal inhibitory concentration of resazurin for multiple N. gonorrhoeae clinical isolates was significantly higher under low oxygen. This effect was specific to resazurin as N. gonorrhoeae was equally susceptible to other antibiotics under low and atmospheric oxygen conditions. The reduced susceptibility of N. gonorrhoeae to resazurin under low oxygen was largely attributed to reduced oxidative stress, as the addition of antioxidants under atmospheric oxygen mimicked the reduced susceptibility to resazurin observed under low oxygen. Together, these data suggest oxygen concentration is an important factor to consider when evaluating the efficacy of new antibiotics against N. gonorrhoeae in vitro. [ABSTRACT FROM AUTHOR]

Primarily owing to the pronounced fluorescence exhibited by its reduced form, resazurin (also known as alamarBlue®) is widely employed as a redox sensor to assess cell viability in in vitrostudies. In an effort to broaden its applicability for in vivo studies, molecular adjustments are necessary to align optical properties with the near-infrared imaging window while preserving redox properties. This study delves into the theoretical characterisation of a set of fluorinated resazurin derivatives proposed by Kachur et al., 2015 examining the influence of fluorination on structural and electrochemical properties. Assuming that the conductor-like polarisable continuum model mimics the solvent effect, the density functional level of theory combining M06-2X/6-311G* was used to calculate the redox potentials. Furthermore, (TD-)DFT calculations were performed with PBE0/def2-TZVP to evaluate nucleophilic characteristics, transition states for fluorination, relative energies, and fluorescence spectra. With the aim of exploring the potential of resazurin fluorinated derivatives as redox sensors tailored for in vivo applications, acid–base properties and partition coefficients were calculated. The theoretical characterisation has demonstrated its potential for designing novel molecules based on fundamental principles. [ABSTRACT FROM AUTHOR]

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Hydrogels were prepared by Steglich esterification and by crosslinking pre-synthesized poly(sorbitol adipate)-graft-poly(ethylene glycol) mono methyl ether (PSA-g-mPEG) using different-chain-length-based disuccinyl PEG. PSA and PSA-g-mPEG were investigated for polymer degradation as a function of time at different temperatures. PSA-g-mPEG hydrogels were then evaluated for their most crucial properties of swelling that rendered them suitable for many pharmaceutical and biomedical applications. Hydrogels were also examined for their Sol-Gel content in order to investigate the degree of cross-linking. Physical structural parameters of the hydrogels were theoretically estimated using the modified Flory–Rehner theory to obtain approximate values of polymer volume fraction, the molecular weight between two crosslinks, and the mesh size of the hydrogels. X-ray diffraction was conducted to detect the presence or absence of crystalline regions in the hydrogels. PSA-g-mPEG hydrogels were then extensively examined for higher and lower molecular weight solute release through analysis by fluorescence spectroscopy. Finally, the cytotoxicity of the hydrogels was also investigated using a resazurin reduction assay. Experimental results show that PSA-g-mPEG provides an option as a biocompatible polymer to be used for pharmaceutical applications. [ABSTRACT FROM AUTHOR]

Resazurin (Alamar Blue, RZ) is a widely utilized fluorescent probe for biological sensing, whose fluorescent intensity can be modulated by changing its redox states; thereby, electrochemical reactivity of RZ is of significance when designing a sensing assay. Herein, we report novel two-way electrochemical reactivity modulation of RZ using host-guest complexation with rigid molecular containers cucurbit[n]uril (CBn, n = 7, 8). The complexation between CBn and RZ is confirmed by 1H NMR measurements and supported by computational simulation, and the binding constants are determined via UV-vis titration. Notably, the voltametric data highlights that the redox reactivity of RZ can be activated or deactivated upon encapsulation by CB8 or CB7, respectively. This two-way reactivity modulation is hypothesized to be mediated by the difference in cavity volume that favors or hinders the approach of water molecules to the encapsulated reaction center during the reduction process. Despite the similar cavity size to CB, molecular containers such as cyclodextrins (CDs) exhibit considerably weaker modulation effects. Our approach can potentially be applied to other redox processes that involve proton transfer, and open new possibilities in supramolecular electrochemistry. [ABSTRACT FROM AUTHOR]

Abstract Background Helminth infections are an important public health problem in humans and have an even greater impact on domestic animal and livestock welfare. Current readouts for anthelmintic drug screening assays are stage development, migration, or motility that can be subjective, laborious, and low in throughput. The aim of this study was to apply and optimize a fluorometric technique using resazurin for evaluating changes in the metabolic activity of Ascaris suum third-stage larvae (L3), a parasite of high economic relevance in swine. Methods Ascaris suum L3 were mechanically hatched from 6- to 8-week embryonated and sucrose-gradient-enriched eggs. Resazurin dye and A. suum L3 were titrated in 96-well microtiter plates, and resazurin reduction activity was assessed by fluorometry after 24 h of incubation. Fluorescence microscopy was used to localize the resazurin reduction site within the larvae. Finally, we exposed A. suum L3 to various stress conditions including heat, methanol, and anthelmintics, and investigated their impact on larval metabolism through resazurin reduction activity. Results We show that the non-fluorescent dye resazurin is reduced inside vital A. suum L3 to fluorescent resorufin and released into the culture media. Optimal assay parameters are 100–1000 L3 per well, a resazurin concentration of 7.5 µg/ml, and incubation at 37 °C/5% CO2 for 24 h. An intact L2 sheath around the L3 of A. suum completely prevents the uptake of resazurin, while in unsheathed L3, the most intense fluorescence signal is observed along the larval midgut. L3 exposed to methanol or heat show a gradually decreased resazurin reduction activity. In addition, 24 h exposure to ivermectin at 0.625 µM, mebendazole at 5 µM, and thiabendazole from 10 to 100 µM significantly decreased larval metabolic activity by 55%, 73%, and 70% to 89%, respectively. Conclusions Together, our results show that both metabolic stressors and anthelmintic drugs significantly and reproducibly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy-to-use method to evaluate metabolic activity of A. suum L3 in vitro. Graphical Abstract

Resazurin (Alamar Blue, RZ) is a widely utilized fluorescent probe for biological sensing, whose fluorescent intensity can be modulated by changing its redox states; thereby, electrochemical reactivity of RZ is of significance when designing a sensing assay. Herein, we report novel two-way electrochemical reactivity modulation of RZ using host-guest complexation with rigid molecular containers cucurbit[n]uril (CBn, n = 7, 8). The complexation between CBn and RZ is confirmed by 1H NMR measurements and supported by computational simulation, and the binding constants are determined via UV-vis titration. Notably, the voltametric data highlights that the redox reactivity of RZ can be activated or deactivated upon encapsulation by CB8 or CB7, respectively. This two-way reactivity modulation is hypothesized to be mediated by the difference in cavity volume that favors or hinders the approach of water molecules to the encapsulated reaction center during the reduction process. Despite the similar cavity size to CB, molecular containers such as cyclodextrins (CDs) exhibit considerably weaker modulation effects. Our approach can potentially be applied to other redox processes that involve proton transfer, and open new possibilities in supramolecular electrochemistry.

With the rise of antibiotic resistance, the drive to discover novel antimicrobial substances and standard testing methods with the aim of controlling transmissive diseases are substantially high. In healthcare sectors and industries, although methods for testing antibiotics and other aqueous-based reagents are well established, methods for testing nanomaterials, non-polar and other particle-based suspensions are still debatable. Hence, utilities of ISO standard validations of such substances have been recalled where corrective actions had to be taken. This paper reports a serial analysis obtained from testing the antimicrobial activities of 10 metallic-based nanomaterials against 10 different pathogens using five different in vitro assays, where the technique, limitation and robustness of each method were evaluated. To confirm antimicrobial activities of metallic-based nanomaterial suspensions, it was found that at least two methods must be used, one being the agar well diffusion method, which was found to be the most reliable method. The agar well diffusion method provided not only information on antimicrobial efficacy through the size of the inhibitory zones, but it also identified antimicrobial ions and synergistic effects released by the test materials. To ascertain the effective inhibitory concentration of nanoparticles, the resazurin broth dilution method is recommended, as MIC can be determined visually without utilising any equipment. This method also overcomes the limit of detection (LoD) and absorbance interference issues, which are often found in the overexpression of cell debris and nanoparticles or quantum dots with optical profiles. In this study, bimetallic AgCu was found to be the most effective antimicrobial nanoparticle tested against across the bacterial (MIC 7 µg/mL) and fungal (MIC 62.5 µg/mL) species. [ABSTRACT FROM AUTHOR]

Germination capacity is an important parameter of seed viability. The direct germination test destroys the seed. Viability evaluation by nondestructive tests allows the saving of seeds for storage and future use. However, predictive values of most nondestructive tests have not been adequately determined. This study investigated performance parameters and optimized assay conditions for a nondestructive colorimetric resazurin seed-viability test of flax seeds (Linum usitatissimum L.). In this study, 800 seeds were tested by resazurin test before germination. Optimization of resazurin test conditions and cutoff value was determined via a "receiver operating characteristic curve" analysis. We found that the optimal incubation time was 2 h. We proposed to measure sample absorption at 595 nm for better seed-viability estimation by resazurin test due to spectral properties of dye. We improved sensitivity of the resazurin test by using measurements adjusted to blank absorption at 595 nm (sensitivity 91%) or to sample absorption at 490 nm (sensitivity 95% versus 81% before corrections). The resazurin test reliably predicted germinable seeds in 72–73% cases and ungerminable seeds in 92–95% cases, with a sensitivity of 91–95% and a specificity of 71–74%. Therefore, the easy and low-cost resazurin test exhibited analytical performance similar to more expensive instrumental tests and can be suitable for nondestructive seed-viability estimation. [ABSTRACT FROM AUTHOR]

Many sophisticated water treatment plants need a reliable, fast, and economical microbial load detection method. We refined a colorimetric assay using the redox dye resazurin to assess viable microorganisms. Here, we have used a mixed bacterial suspension of significant multi-drug-resistant coliform bacteria isolated from hospital wastewater and constructed a resazurin reduction calibration curve which could accurately predict the level of microbial contamination. The number of viable microorganisms was calculated from calibration curve in terms of log colony forming units (CFU) per milliliter. Ultrasonication disinfection of bacterial suspension for a duration of 50 min measured by resazurin assay depicted a reduction of 16.94%, 26.48%, and 37.69% at 410 W, 580 W, and 700 W, respectively. A synergistic effect of the combined methods of ultrasonication and heat disinfection treatments on raw wastewater and secondary wastewater effluent was observed and was also evaluated using both resazurin assay and standard plate count method. For raw wastewater, about 1.8 log reduction was observed for ultrasonication alone and 4 log CFU/mL reduction for thermosonication. In the secondary wastewater effluent, a reduction of 2.9 and 3.2 log CFU/mL was recorded for ultrasonication and thermosonication respectively. Resazurin microbial viability test results were highly comparable with conventional colony plate count for all treatment procedures, suggesting its appropriateness for quick and reliable wastewater sample microbial viability monitoring. [ABSTRACT FROM AUTHOR]

The time-consuming nature of current methods for detecting antimicrobial resistance (AMR) to guide mastitis treatment and for surveillance, drives innovation towards faster, easier, and more portable technology. Rapid on-farm testing could guide antibiotic selection, reducing misuse that contributes to resistance. We identify challenges that arise when developing miniaturized antibiotic susceptibility tests (AST) for rapid on-farm use directly in milk. We experimentally studied three factors: sample matrix (specifically milk or spoiled milk); the commensal bacteria found in fresh bovine milk; and result time on the performance of miniaturised AST. Microfluidic "dip-and-test" devices made from microcapillary film (MCF) were able to monitor Gram-negative bacterial growth colourimetrically even in the presence of milk and yoghurt (used to simulate spoiled milk samples), as long as this sample matrix was diluted 1:5 or more in growth medium. Growth detection kinetics using resazurin was not changed by milk at final concentrations of 20% or lower, but a significant delay was seen with yoghurt above 10%. The minimum inhibitory concentration (MIC) for ciprofloxacin and gentamicin was increased in the presence of higher concentrations of milk and yoghurt. When diluted to 1% all observed MIC were within range, indicating dilution may be sufficient to avoid milk matrix interfering with microfluidic AST. We found a median commensal cell count of 6 × 105 CFU/mL across 40 healthy milk samples and tested if these bacteria could alter microfluidic AST. We found that false susceptibility may be observed at early endpoint times if testing some pathogen and commensal mixtures. However, such errors are only expected to occur when a susceptible commensal organism is present at higher cell density relative to the resistant pathogen, and this can be avoided by reading at later endpoints, leading to a trade-off between accuracy and time-to-result. We conclude that with further optimisation, and additional studies of Gram-positive organisms, it should be possible to obtain rapid results for microfluidic AST, but a trade-off is needed between time-to-result, sample dilution, and accuracy. [ABSTRACT FROM AUTHOR]

Neisseria gonorrhoeae has developed resistance to every antibiotic currently approved for the treatment of gonorrhea, prompting the development of new therapies. The phenoxazine dye resazurin exhibits robust antimicrobial activity against N. gonorrhoeae in vitro but fails to limit vaginal colonization by N. gonorrhoeae in a mouse model. The lack of in vivo efficacy may be due to oxygen limitation as in vitro susceptibility assays with resazurin are conducted under atmospheric oxygen while a microaerophilic environment is present in the vagina. Here, we utilized broth microdilution assays to determine the susceptibility of N. gonorrhoeae to resazurin under low and atmospheric oxygen conditions. The minimal inhibitory concentration of resazurin for multiple N. gonorrhoeae clinical isolates was significantly higher under low oxygen. This effect was specific to resazurin as N. gonorrhoeae was equally susceptible to other antibiotics under low and atmospheric oxygen conditions. The reduced susceptibility of N. gonorrhoeae to resazurin under low oxygen was largely attributed to reduced oxidative stress, as the addition of antioxidants under atmospheric oxygen mimicked the reduced susceptibility to resazurin observed under low oxygen. Together, these data suggest oxygen concentration is an important factor to consider when evaluating the efficacy of new antibiotics against N. gonorrhoeae in vitro.

The resazurin reduction test is one of the basic tests for bacterial culture viability and drug resistance endorsed by the World Health Organisation. At the same time, conventional spectrophotometric and spectrofluorimetric methods demand rather bulky and expensive equipment. This induces a challenge for developing simpler approaches to sensor systems that are portable and applicable in resource-limited settings. In this work, we address two such alternative approaches, based on the colour processing of the microbiological plate's photographic images and single-channel photometry with a recently developed portable microbiological analyser. The key results consist of establishing a sequential linear correspondence between the concentration of resorufin produced due to the reduction of resazurin by viable bacteria as determined by the UV-Vis studies, the intensity of the a* channel of the CIE L*a*b* colour space and the transmitted light intensity registered by a luxmeter under the LED illumination with a yellow colour filter. This route is illustrated with the chemical system "Hydrazine hydrate – resazurin", isolating the target colour change-inducing reaction and the test of determining the minimal inhibition concentration of the antibacterial first-line drug isoniazid acting on the culture of the H37Rv strain of M. tuberculosis. [ABSTRACT FROM AUTHOR]

Background: Helminth infections are an important public health problem in humans and have an even greater impact on domestic animal and livestock welfare. Current readouts for anthelmintic drug screening assays are stage development, migration, or motility that can be subjective, laborious, and low in throughput. The aim of this study was to apply and optimize a fluorometric technique using resazurin for evaluating changes in the metabolic activity of Ascaris suum third-stage larvae (L3), a parasite of high economic relevance in swine. Methods: Ascaris suum L3 were mechanically hatched from 6- to 8-week embryonated and sucrose-gradient-enriched eggs. Resazurin dye and A. suum L3 were titrated in 96-well microtiter plates, and resazurin reduction activity was assessed by fluorometry after 24 h of incubation. Fluorescence microscopy was used to localize the resazurin reduction site within the larvae. Finally, we exposed A. suum L3 to various stress conditions including heat, methanol, and anthelmintics, and investigated their impact on larval metabolism through resazurin reduction activity. Results: We show that the non-fluorescent dye resazurin is reduced inside vital A. suum L3 to fluorescent resorufin and released into the culture media. Optimal assay parameters are 100–1000 L3 per well, a resazurin concentration of 7.5 µg/ml, and incubation at 37 °C/5% CO2 for 24 h. An intact L2 sheath around the L3 of A. suum completely prevents the uptake of resazurin, while in unsheathed L3, the most intense fluorescence signal is observed along the larval midgut. L3 exposed to methanol or heat show a gradually decreased resazurin reduction activity. In addition, 24 h exposure to ivermectin at 0.625 µM, mebendazole at 5 µM, and thiabendazole from 10 to 100 µM significantly decreased larval metabolic activity by 55%, 73%, and 70% to 89%, respectively. Conclusions: Together, our results show that both metabolic stressors and anthelmintic drugs significantly and reproducibly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy-to-use method to evaluate metabolic activity of A. suum L3 in vitro. [ABSTRACT FROM AUTHOR]

Beer refermentation in bottles is an industrial process utilized by breweries where yeast and fermentable extract are added to green beer. The beer is refermented for a minimum of 2 weeks before distribution, with the physiological state of the yeast a critical factor for successful refermentation. Ideally, fresh yeast that is propagated from a dedicated propagation plant should be used for refermentation in bottles. Here, we explored the applicability of the fluorescent and redox‐sensitive dye, resazurin, to assess cellular metabolism in yeast and its ability to differentiate between growth stages. We applied this assay, with other markers of yeast physiology, to evaluate yeast quality during a full‐scale industrial propagation. Resazurin allowed the discrimination between the different growth phases in yeast and afforded a more in‐depth understanding of yeast metabolism during propagation. This assay can be used to optimize the yeast propagation process and cropping time to improve beer quality. Take‐away: Resazurin can discriminate between different growth phases in yeast.Resazurin permits deeper understanding of yeast physiology during a brewery propagation.Yeast metabolic activity was decreased at the end of an industrial‐scale propagation. [ABSTRACT FROM AUTHOR]

Knowledge of microbial functioning linked to dissolved organic matter (DOM) in hypersaline lakes is limited. This study aimed to understand this relationship in surface and subsurface sediments and how it is affected by daytime and night‐time variability.A sampling regime, including daytime and night‐time measurements, was designed to record aerobic respiration, extracellular enzyme activity (β‐glucosidase, leu‐aminopeptidase and phosphatase), prokaryotic cell density and viability, content of pigment and extracellular polymeric substances (EPS), and organic matter weight in the surface and subsurface sediments. The main physicochemical characteristics and optical properties of DOM in the surface and interstitial waters were determined.Larger differences were detected in biofilm microbial activity between surface and subsurface sediments than between day and night, as a result of higher prokaryotic cell density and higher availability of fresh DOM at the surface, possibly released from primary production. Redox conditions, pH and DOM quantity and quality were strongly correlated with enzyme activity and variability in respiration, and might determine (through modifying nitrogen (N) and phosphorus (P) availability) how microbes use organic compounds differently at the two sediment depths.Low prokaryotic viability indicated that the microbial community was stressed in both surface and subsurface sediments. By contrast, higher EPS and pigment content and lower metabolic efficiency at the surface layer indicated harsher conditions compared to the subsurface layer. Pigment presence and variation in daily oxygen levels in the subsurface layer indicate that the two sediment depths are interconnected.Results suggest that surface and subsurface microbes have a distinct role in hypersaline shallow lake sediment. Microorganisms in surface sediments form a structured mat with high EPS and carotenoids and enhanced carbon metabolism. The mat also serves as a protection shield from light radiation, salinity and desiccation. At the subsurface, the lower availability of fresh organic compounds and oxygen reduces microbial biomass and activity which is, in turn, more efficient and focused on N and P acquisition. [ABSTRACT FROM AUTHOR]

Cell viability and metabolic activity are ubiquitous parameters used in biochemistry, molecular biology, and biotechnological studies. Virtually all toxicology and pharmacological projects include at some point the evaluation of cell viability and/or metabolic activity. Among the methods used to address cell metabolic activity, resazurin reduction is probably the most common. At variance with resazurin, resorufin is intrinsically fluorescent, which simplifies its detection. Resazurin conversion to resorufin in the presence of cells is used as a reporter of metabolic activity of cells and can be detected by a simple fluorometric assay. UV–Vis absorbance is an alternative technique but is not as sensitive. In contrast to its wide empirical "black box" use, the chemical and cell biology fundamentals of the resazurin assay are underexplored. Resorufin is further converted to other species, which jeopardizes the linearity of the assays, and the interference of extracellular processes has to be accounted for when quantitative bioassays are aimed at. In this work, we revisit the fundamentals of metabolic activity assays based on the reduction of resazurin. Deviation to linearity both in calibration and kinetics, as well as the existence of competing reactions for resazurin and resorufin and their impact on the outcome of the assay, are addressed. In brief, fluorometric ratio assays using low resazurin concentrations obtained from data collected at short time intervals are proposed to ensure reliable conclusions. [ABSTRACT FROM AUTHOR]

Biopolymeric films were prepared with gelatin, plasticizer, and three different types of antioxidants (ascorbic acid, phytic acid, and BHA) corresponding to different mechanisms in activity. The antioxidant activity of films was monitored for 14 storage days upon color changes using a pH indicator (resazurin). The instant antioxidant activity of films was measured by a DPPH free radical test. The system using resazurin was composed of an agar, an emulsifier, and soybean oil to simulate a highly oxidative oil-based food system (AES-R). Gelatin-based films (GBF) containing phytic acid showed higher tensile strength and energy to break than all other samples due to the increased intermolecular interactions between phytic acid and gelatin molecules. The oxygen barrier properties of GBF films containing ascorbic acid and phytic acid increased due to the increased polarity, while GBF films containing BHA showed increased oxygen permeability compared to the control. According to "a-value" (redness) of the AES-R system tested with films, films incorporating BHA showed the most retardation of lipid oxidation in the system. This retardation corresponds to 59.8% antioxidation activity at 14 days, compared with the control. Phytic acid-based films did not show antioxidant activity, whereas ascorbic acid-based GBFs accelerated the oxidation process due to its prooxidant activity. The comparison between the DPPH free radical test and the control showed that the ascorbic acid and BHA-based GBFs showed highly effective free radical scavenging behavior (71.7% and 41.7%, respectively). This novel method using a pH indicator system can potentially determine the antioxidation activity of biopolymer films and film-based samples in a food system. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance remains a global threat with ~ 5 million deaths in 2019 alone and 10 million deaths projected every year by 2050. Current tools employed in the analysis of bacteria can be time inefficient, leading to delayed diagnosis and treatment. In this work, we develop a microfluidic setup capable of bacteria incubation and detection of growth in ~ 2 h. We fabricated polydimethylsiloxane (PDMS) microchips via soft lithography, enclosed microchannels by plasma bonding to glass, and utilized PDMS blocks for simplified connection of devices to a flow system. We generated uniform droplets enclosing zero, one or two bacteria within our devices, and incubated droplet-encapsulated bacteria with 100 × lower concentrations of a fluorescence probe of bacterial growth compared to prior work. We assessed bacterial growth via laser induced fluorescence after room temperature incubation for 2 h and obtained a range of signals corresponding to droplets with or without bacteria. Our devices allow for online droplet incubation, monitoring, detection, and tracking. Developing microfluidic chips for single bacteria studies will improve the analysis and treatment of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Background Vancomycin and linezolid resistance among enterococci is an increasing problem due to a lack of alternative antibiotics. Early identification of vancomycin-resistant and linezolid-resistant strains can help prevent the spread of resistance to these antibiotics. Hence, early, rapid and accurate detection of vancomycin and linezolid resistance is critical. Objectives The resazurin microplate method (RMM) was developed for detecting vancomycin and linezolid susceptibility among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) clinical isolates, and its performance was further evaluated. Methods A total of 209 non-duplicate clinical isolates and three strains from the faeces of domestic animals, including 142 E. faecalis (71 linezolid non-susceptible and 71 linezolid susceptible) and 70 E. faecium (23 vancomycin non-susceptible, 23 vancomycin susceptible, 12 linezolid non-susceptible and 12 linezolid susceptible), were tested using RMM. Results The susceptibility of E. faecium to vancomycin was detected within 5 h, with high susceptibility (23/23) and specificity (23/23). The susceptibility of E. faecalis and E. faecium to linezolid was detected within 4 h, with specificities of 98.59% and 100% and susceptibilities of 94.37% and 58.33% for E. faecalis and E. faecium , respectively. Conclusions RMM had a good positive predictive value for the detection of vancomycin-non-susceptible E. faecium and linezolid-non-susceptible E. faecalis. It thus has the potential to become an alternative method for the rapid screening of these resistant pathogens in clinical practice. [ABSTRACT FROM AUTHOR]

Cancer is a global medical problem and, despite research efforts in the field of tumor treatment, there is currently a shortage of specific anticancer drugs. Most anticancer drugs show significant side effects. The liver is the organ that has central functions in drug metabolism, being a major target of the harmful action of anticancer compounds. In this context, it is essential to evaluate the cytotoxic effects of potential anticancer substances. Therefore, hepatotoxicity and hepatocyte viability were determined in vitro to evaluate the action of seven new local thiosemicarbazide coordination compounds (CCT) on normal liver cells. Doxorubicin was used as a reference substance. The control group consisted of hepatocytes not exposed to CCT action. The cell viability of hepatocytes treated with CCT decreased significantly by 5–12% compared to the control, but was statistically significantly higher by 5–14% compared to doxorubicin, except after CMD-8 and CMT-67 influence, when it does not change. Thus, new local CCT had a selective effect on hepatocytes in vitro and were less hepatotoxic compared to doxorubicin, which may be the basis for further study of its potential in anticancer drugs. [ABSTRACT FROM AUTHOR]

Objectives: Actinomycetoma is a chronic granulomatous disease affecting skin, subcutaneous tissue, fascia, muscle and bones. With increasing resistance against commonly used treatment regimens, susceptibility testing is urgently needed.Methods: We developed an in vitro susceptibility assay for Actinomadura madurae, one of the common causative agents of actinomycetoma, employing resazurin for endpoint reading. Using this assay, reproducible MICs were determined for the most commonly used antibacterial agents for actinomycetoma treatment. The tested antibacterial agents included trimethoprim/sulfamethoxazole, amikacin, streptomycin, amoxicillin, ceftriaxone, gentamicin, ciprofloxacin, doxycycline, imipenem, linezolid, penicillin G and rifampicin.Results: Following the clinical breakpoints as stated by CLSI, 100% of the tested strains were susceptible to trimethoprim/sulfamethoxazole (MIC 0.03/0.59-1/19 mg/L), amikacin (MIC 0.0078-0.25 mg/L), doxycycline (MIC <0.25-1 mg/L) and linezolid (MIC <0.25-2 mg/L), 90% to ciprofloxacin (MIC <0.25-2 mg/L), 80% to ceftriaxone (MIC <0.5 to >64 mg/L) and imipenem (MIC <0.25-32 mg/L) and only 20% to amoxicillin (MIC <0.5 to >64 mg/L) and rifampicin (MIC 0.5 to >32 mg/L).Conclusions: Determinations of MICs by visual readings of colour changes versus spectrophotometric readings were comparable. This convenient visual reading has the advantage of feasible implementation in endemic settings. [ABSTRACT FROM AUTHOR]

Sr-hydroxyapatite has drawn much attention with promising applications in biomedical engineering especially in drug delivery and tissue repair. A novel protocol for the preparation of Strontium hydroxyapatite (Sr-HAP) and Strontium hydroxyapatite encapsulated by graphitic carbon nitride (Sr-HAP@gCN) with high purity and better homogeneity was adopted for potential use in orthopaedic surgeries. The physiological pathway of Strontium and calcium inside humans follow the same trend leading to the mineral deposition of the bone. In the present study, a novel attempt in synthesizing Sr-HAP and Sr-HAP@gCN by the hydrothermal method is proposed for obtaining an ideal biomaterial with potential biocompatibility and pharmacological activity. Phase identification by X-ray diffraction (XRD), Fourier transform Infra-Red spectroscopic analysis (FTIR), Electron & atomic force microscopic studies, Thermogravimetric and Raman spectroscopic analysis was performed on the prepared Sr-HAP and Sr-HAP@gCN to characterize the incorporation efficiency of Strontium. Resazurin microtiter assay and MTT assay were used to evaluate antimicrobial susceptibility and biocompatibility. The typical crystal phase of Sr-HAP and a phase change in Sr-HAP@gCN have been demonstrated with special reference to the interfacial connection between gCN and Sr-HAP through FT-IR studies. Raman spectroscopy revealed the archetypal signature of symmetric and asymmetric P O vibrations pertaining to linearity change with the addition of Strontium. The biological effect of Sr-HAP and Sr-HAP@gCN was quite significant in inducing mineralization when kept immersed in simulated body fluid (SBF) for 21 days. Promising results pertaining to antimicrobial susceptibility and biocompatibility have been proved as an encouraging choice for drug delivery applications and bone filling applications. [Display omitted] • Sr-HAP@gCN nanocomposite was successfully synthesized by hydrothermal method • A low surface roughness value was observed for Sr-HAP@gCN nanocomposite • Sr-HAP@gCN nanocomposite showed significant antibacterial effect and cell viability [ABSTRACT FROM AUTHOR]

[Display omitted] • Antimicrobial activity of different photocatalytic surfaces is tested via resazurin assay. • The inoculated hydrogel carriers are anchored onto the studied surfaces. • The four different situations are examined; irradiated photocatalytic surface and reference; photocatalytic surface and reference in dark. • Reduction of total analysis time; better recovery results, and the applicability to different surfaces is demonstrated comparing with standard process. The antimicrobial activity of different types of photocatalytic surfaces (titania porous and nonporous phthalocyanine films) was tested using a resazurin assay. The gram-negative bacteria Escherichia coli was chosen as the model microorganism. A bacteria inoculum with a concentration of 1.75 × 106 CFU/ml was applied to hydrogel carriers and these were subsequently placed on the study surfaces. Half of the samples were kept in the dark and the other half were illuminated. After 4 h, the carriers were transferred to vials, resazurin added and the number of viable cells in the mixture counted. For nonporous surfaces, the effect of the addition of a biocide was investigated. The authors observed that as the amount of biocide in the layer increased, the proportion of viable cells in the samples kept in the dark decreased. These results are in good agreement with general assumptions. For the titania surfaces, the antimicrobial activity (characterized by ΔR) obtained through resazurin assay was compared to that obtained through the use of standard techniques. The final activity was very similar (the ΔR from the resazurin assay was 1.54 and the standard test gave a result of 1.56). The results confirm the reliability of the proposed method, and the use of carriers give us the possibility to test much larger installed surfaces without a need to cause damage to them. [ABSTRACT FROM AUTHOR]

With the rise of antibiotic resistance, the drive to discover novel antimicrobial substances and standard testing methods with the aim of controlling transmissive diseases are substantially high. In healthcare sectors and industries, although methods for testing antibiotics and other aqueous-based reagents are well established, methods for testing nanomaterials, non-polar and other particle-based suspensions are still debatable. Hence, utilities of ISO standard validations of such substances have been recalled where corrective actions had to be taken. This paper reports a serial analysis obtained from testing the antimicrobial activities of 10 metallic-based nanomaterials against 10 different pathogens using five different in vitro assays, where the technique, limitation and robustness of each method were evaluated. To confirm antimicrobial activities of metallic-based nanomaterial suspensions, it was found that at least two methods must be used, one being the agar well diffusion method, which was found to be the most reliable method. The agar well diffusion method provided not only information on antimicrobial efficacy through the size of the inhibitory zones, but it also identified antimicrobial ions and synergistic effects released by the test materials. To ascertain the effective inhibitory concentration of nanoparticles, the resazurin broth dilution method is recommended, as MIC can be determined visually without utilising any equipment. This method also overcomes the limit of detection (LoD) and absorbance interference issues, which are often found in the overexpression of cell debris and nanoparticles or quantum dots with optical profiles. In this study, bimetallic AgCu was found to be the most effective antimicrobial nanoparticle tested against across the bacterial (MIC 7 µg/mL) and fungal (MIC 62.5 µg/mL) species.

Abstract Background Nocardia species are ubiquitous in natural environments and can cause nocardiosis. In the present study, the use of Resazurin salt and Spectrophotometry were proposed as alternative methods to reduce subjectivity in the interpretation of susceptibility results to antimicrobials by the broth microdilution method for Nocardia spp. Results The susceptibility of Nocardia spp. isolates to Amikacin, Ciprofloxacin, Minocycline and Trimethoprim-Sulfamethoxazole was evaluated by Minimum Inhibitory Concentration (MIC) determinations by the broth microdilution method. To verify cellular growth, the colour-changing dye Resazurin was applied, the Optical Densities were measured on a spectrophotometer, and both were compared to Clinical and Laboratory Standards Institute (CLSI) Gold Standard method (visual MIC determination). Percentages of essential and categorical agreements and interpretative categorical errors were calculated within each method (intra-reading) and between them (inter-reading). The Gold Standard visual reading demonstrated 100% of essential and categorical intra-reading agreements for Amikacin, and there was no error when compared with the alternative methods. For Ciprofloxacin, the comparison between the Gold Standard and the Spectrophotometric reading showed 91.5% of essential agreement. In the categorical intra-reading analysis for Minocycline, there were 88.1 and 91.7% in the Gold Standard and in the Spectrophotometric readings, respectively, and 86.4% of concordance between them. High rates of categorical agreement were also observed on the Trimethoprim-Sulfamethoxazole analyses, with 93.7% for the Gold Standard, 84.9% for the Resazurin readings, and 80.5% between them. Conclusions The alternative methods with Resazurin and Spectrophotometric readings showed high agreement rates with the Gold Standard.

Disc and well diffusion methods were used to test the antibacterial activity of methanol extract and ethanol extract of the selected ethnomedicinal plant viz. Garcinia pedunculata Roxb. ex Buch.-Ham. Both the methanol extract and ethanol extract were subjected to antibacterial activity assay against the six clinical isolates. Antibiotic sensitivity test of the test bacteria against standard antibiotics were also determined. All the bacterial pathogens (Staphylococcus aureus, Enterococcus faecalis, Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) were exposed to the plant extract in triplicates. The investigation revealed the potency of G. pedunculata extract as an effective antibacterial agent against both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB). G. pedunculata are evaluated to be bactericidal against the tested bacteria. The antibacterial activity may be due to an individual compound or synergistic effect of more than one compound present in the medicinal plant extract. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the plant extracts were also investigated by using the broth microdilution method. HPLC-MS of the methanol extract of G. pedunculata fruit revealed the presence of Hydroxy Citric Acid Lactone (MW-190), Garcinone-E (MW-464), a-Mangostin (MW-410), β–Mangostin (MW-424), and γ-Mangostin (MW-396).

Many studies in ecohydrology focusing on hydrologic transport argue that longer residence times across a stream ecosystem should consistently result in higher biological uptake of carbon, nutrients, and oxygen. This consideration does not incorporate the potential for biologically mediated reactions to be limited by stoichiometric imbalances. Based on the relevance and co-dependences between hydrologic exchange, stoichiometry, and biological uptake, and acknowledging the limited amount of field studies available to determine their net effects on the retention and export of resources, we quantified how microbial respiration is controlled by the interactions and supply of essential nutrients needed (C, N, P) in a headwater stream in Colorado, USA. For this, we conducted two rounds of nutrient experiments, each consisting of four sets of continuous injections of Cl- as a conservative tracer, resazurin as a proxy for aerobic respiration, and one of the following nutrient treatments: a) N, b) N+C, c) N+P, and d) C+N+P. Nutrient treatments were considered as known system modifications to alter metabolism, and statistical tests helped identify the relationships between hydrologic transport and respiration metrics. We found that as discharge changed significantly between rounds and across stoichiometric treatments, a) transient storage mainly occurred in side pools along the main channel and was proportional to discharge, and b) microbial respiration remained similar between rounds and across stoichiometric treatments. Together, our results indicate that residence time alone could be a weak predictor of stream respiration due to the relevance of local and dynamic variations in stoichiometric conditions. [ABSTRACT FROM AUTHOR]

The resazurin (raz)‐resorufin (rru) smart tracer system has led to significant advances in hydrologic and biogeochemical sciences but its application has been limited in scope and geography by the short duration that it has been assumed samples can be reliably stored before analysis. For the first time, we quantify the effects of sample storage duration and temperature on measured raz and rru concentrations in order to identify robust storage protocols. Raz/rru concentrations equivalent to those typically used in field and laboratory applications were prepared in three different mediums (deionized water, groundwater, and surface water). Samples were stored at 20°C, 5°C, and −20°C, representing average room, refrigerator, and freezer temperatures. Analysis of raz/rru concentration changes were conducted systematically during an 84‐day period, with higher frequency of analysis in the first 7 d. For samples stored at room temperature, changes of up to 22.6% were observed over the initial 24 h. In contrast, for samples stored in the refrigerator and freezer, changes of up to 8.4% and 6.9% were observed in the same period, respectively. Freezing samples provided the best preservation, yielding a maximum of ~ 10% change after 14 d compared to a maximum of ~ 30% change when cooled. It is generally recommended that raz/rru samples be stored cooled for up to 48 h and frozen for up to 14 d. This offers exciting opportunities to broaden the application of the raz‐rru system to undertake measurements in wider geographical (remote) locations and at increased sampling frequencies. [ABSTRACT FROM AUTHOR]

Objectives: Diagnosis of tuberculosis (TB) is challenging, especially in a resource-limited setting. Noncommercial alternatives such as colorimetric assays must be used for effective diagnosis and prompt treatment. In this study we aimed to determine the diagnostic performance, time to detection, cost, contamination rate, and ease of performance of the oxidation-reduction assays; Resazurin tube assay (RETA) and Malachite Green decolorization assay (MGDA) and their modifications using para-nitro benzoic acid (PNB) for detection of TB against the results on Lowenstein-Jensen medium (LJ) medium. Methods: Two-hundred-seventeen samples were subjected to colorimetric assays by incubating the inoculated media at 370C. Dyes were added on days 10, 14, 18, 28, and 42 days of incubation, and color change was noted after 24 hours and was compared with results on the LJ medium. Results: Diagnostic performance of colorimetric assays increased from day 10 to day 28 (maximum at day 28), while it was the same on days 28 and 42. The sensitivity, specificity, and diagnostic accuracy of RETA were 87.6%, 95.7%, and 88.6%, while that of MGDA were 81.7%, 95.5%, and 86.5%, respectively. On day 18, more than 51% of the samples were positive by colorimetric assays, whereas only 20.4% were positive by LJ culture. In addition, the colorimetric assays were as economical as LJ culture. Conclusion: Colorimetric methods can potentially become non-commercial alternatives for rapidly detecting Mycobacterium. tuberculosis, especially in TB laboratories with limited resources. The ease of interpreting the results and its cost-effectiveness is an additional advantage. [ABSTRACT FROM AUTHOR]

Copyright of ANKEM Antibiyotik & Kemoterapi Dergisi is the property of ANKEM Antibiyotik & Kemoterapi Dergisi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The time-consuming nature of current methods for detecting antimicrobial resistance (AMR) to guide mastitis treatment and for surveillance, drives innovation towards faster, easier, and more portable technology. Rapid on-farm testing could guide antibiotic selection, reducing misuse that contributes to resistance. We identify challenges that arise when developing miniaturized antibiotic susceptibility tests (AST) for rapid on-farm use directly in milk. We experimentally studied three factors: sample matrix (specifically milk or spoiled milk); the commensal bacteria found in fresh bovine milk; and result time on the performance of miniaturised AST. Microfluidic “dip-and-test” devices made from microcapillary film (MCF) were able to monitor Gram-negative bacterial growth colourimetrically even in the presence of milk and yoghurt (used to simulate spoiled milk samples), as long as this sample matrix was diluted 1:5 or more in growth medium. Growth detection kinetics using resazurin was not changed by milk at final concentrations of 20% or lower, but a significant delay was seen with yoghurt above 10%. The minimum inhibitory concentration (MIC) for ciprofloxacin and gentamicin was increased in the presence of higher concentrations of milk and yoghurt. When diluted to 1% all observed MIC were within range, indicating dilution may be sufficient to avoid milk matrix interfering with microfluidic AST. We found a median commensal cell count of 6 × 105 CFU/mL across 40 healthy milk samples and tested if these bacteria could alter microfluidic AST. We found that false susceptibility may be observed at early endpoint times if testing some pathogen and commensal mixtures. However, such errors are only expected to occur when a susceptible commensal organism is present at higher cell density relative to the resistant pathogen, and this can be avoided by reading at later endpoints, leading to a trade-off between accuracy and time-to-result. We conclude that with further optimisation, and additional studies of Gram-positive organisms, it should be possible to obtain rapid results for microfluidic AST, but a trade-off is needed between time-to-result, sample dilution, and accuracy.

The resazurin reduction test is one of the basic tests for bacterial culture viability and drug resistance endorsed by the World Health Organisation. At the same time, conventional spectrophotometric and spectrofluorimetric methods demand rather bulky and expensive equipment. This induces a challenge for developing simpler approaches to sensor systems that are portable and applicable in resource-limited settings. In this work, we address two such alternative approaches, based on the colour processing of the microbiological plate’s photographic images and single-channel photometry with a recently developed portable microbiological analyser. The key results consist of establishing a sequential linear correspondence between the concentration of resorufin produced due to the reduction of resazurin by viable bacteria as determined by the UV-Vis studies, the intensity of the a* channel of the CIE L*a*b* colour space and the transmitted light intensity registered by a luxmeter under the LED illumination with a yellow colour filter. This route is illustrated with the chemical system “Hydrazine hydrate – resazurin”, isolating the target colour change-inducing reaction and the test of determining the minimal inhibition concentration of the antibacterial first-line drug isoniazid acting on the culture of the H37Rv strain of M. tuberculosis.

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[Display omitted] • Resazurin was used as a probe for rapid and accurate detection of HOBr in the water environment. • The probe showed excellent selectivity, high sensitivity and fast response for HOBr. • The generation of HOBr in chlorination disinfection and PMS-degradation was successfully tracked. • Test strips were prepared to detect HOBr conveniently and quickly. The highly reactive hypobromous acid (HOBr), which is generated after chlorination process of tap water, acts as a precursor of toxic brominated disinfection by-products (Br-DBPs) and further reacts with organic matter. In addition, HOBr produced from the oxidation of Br− during the degradation of pollutants by peroxymonosulfate (PMS, HSO 5 −) can be considered as the cause of the expedited degradation of pollutants. Therefore, it is particularly important to detect HOBr level in the water environment. Resazurin was selected as a fluorescent probe for selective recognition of HOBr in the water environment. The probe exhibited excellent spectral performance and showed high sensitivity to HOBr (LOD = 515 nM). This method has a relatively ideal recovery rate for HOBr detection in environmental water samples. Furthermore, the HOBr production during the chlorination disinfection process was simulated and the HOBr generated from this process was detected by the probe. Importantly, the process of HOBr recognition by the probe is accompanied by the change of color. Based on this, the relationship between the change of color B/G value and HOBr concentration was successfully constructed. The probe was loaded on the filter paper to make a test strip, which was utilized to the detection of HOBr. Collectively, this work provided a promising and powerful method for HOBr detection in the environment. [ABSTRACT FROM AUTHOR]

ABSTRACT A method to rapidly quantify predatory bacterial cell populations using resazurin reduction to resorufin and its resulting fluorescence kinetics (dF/dt) are described. The reliability of this method to measure the predatory populations was demonstrated with the type strain, Bdellovibrio bacteriovorus HD100, as well as B. bacteriovorus 109J and two natural isolates, Halobacteriovorax strains JA-1 and JA-3, with clear correlation when densities were between 107 and 109 PFU/ml. Resazurin was also used to evaluate how B. bacteriovorus HD100 and Halobacteriovorax strain JA-1 respond to harmful conditions, i.e., exposure to sodium dodecyl sulfate (SDS), with both the dF/dt and PFU/ml indicating Halobacteriovorax strain JA-1 is more sensitive to this surfactant. Tests were also performed using media of different osmolalities, with the dF/dt values matching the 24-h predatory activities reasonably well. Finally, this method was successfully applied in near real-time analyses of predator-prey dynamics and, when coupled with SDS, was capable of differentiating between the predatory and prey populations. All of these tests serve to prove this method is (i) very rapid, needing only 15 min from start to finish; (ii) very reliable with different predatory bacterial species; and (iii) very versatile as it can be easily adapted to measure predatory numbers and activities in a range of experiments. IMPORTANCE Bdellovibrio and like organisms are predatory bacteria that are capable of attacking, killing, and consuming many bacterial pathogens, including multidrug-resistant strains. These qualities have led to them being labeled as “living antibiotics.” Research work with these remarkable strains, however, has been hampered by long growth times needed to quantify the predatory populations through plaque assays, which typically take 4 days to develop. Here, we describe a fluorescence-based method using the conversion of resazurin (low fluorescence) to resorufin (high fluorescence) after it is reduced by the predators’ NADH. Not only do we show that the fluorescence correlates strongly with the predatory concentration and that we can use it to evaluate if the predators are viable, but the entire procedure from start to finish takes only 15 min, drastically reducing the time researchers need to quantify the predatory numbers. Employing this technique will greatly advance research related to predatory bacteria and their potential applications.