RESUMEN
The precision and repeatability of in vivo biological studies is predicated upon methods for isolating a targeted subsystem from external sources of noise and variability. However, in many experimental frameworks, this is made challenging by nonstatic environments during host cell growth, as well as variability introduced by manual sampling and measurement protocols. To address these challenges, we developed Chi.Bio, a parallelised open-source platform that represents a new experimental paradigm in which all measurement and control actions can be applied to a bulk culture in situ. In addition to continuous-culturing capabilities, it incorporates tunable light outputs, spectrometry, and advanced automation features. We demonstrate its application to studies of cell growth and biofilm formation, automated in silico control of optogenetic systems, and readout of multiple orthogonal fluorescent proteins in situ. By integrating precise measurement and actuation hardware into a single low-cost platform, Chi.Bio facilitates novel experimental methods for synthetic, systems, and evolutionary biology and broadens access to cutting-edge research capabilities.
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Reactores Biológicos , Técnicas de Cultivo/instrumentación , Optogenética/instrumentación , Automatización , Biopelículas , Proliferación Celular , Simulación por Computador , Programas InformáticosRESUMEN
The applied surface dose is a key parameter for the measurement of toxic effects of airborne particles by air liquid interface exposure of human lung cells. Besides online measurement of the deposited particle mass by quartz crystal microbalance frequently other dose metrics such as particle size distribution, surface and agglomeration state are required. These particle properties and their spatial distribution can be determined by digital processing of micrographs obtained by transmission electron microscopy (TEM). Here, we report the development and characterization of a novel holder for film coated TEM copper grids, which allows for sampling under identical geometric and ambient conditions as in a cell culture chamber. The sample holder avoids artefacts by reliable grounding of the grids and improves handling of the grids to prevent damage of the sensitive film. This sample holder is applied during exposure experiments with titanium dioxide nanoparticles. The measured dose of 0.2 µg/cm² corresponds well to the mass loading signal of the quartz crystal microbalance. Additionally, the spatial distribution of particles on the sampling surface shows a good homogeneity of deposition. This novel sampling method allows verifying other dosimetry methods and gives additional information about particle properties and homogeneity of the dose.
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Microscopía Electrónica de Transmisión/métodos , Material Particulado/administración & dosificación , Aerosoles/administración & dosificación , Cobre/química , Técnicas de Cultivo/instrumentación , Diseño de Equipo , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Pulmón/citología , Nanopartículas del Metal/administración & dosificación , Microscopía Electrónica de Transmisión/instrumentación , Tamaño de la Partícula , Tecnicas de Microbalanza del Cristal de Cuarzo , Titanio/administración & dosificaciónRESUMEN
This paper presents a new surface modification strategy to develop a poly(ethylene terephthalate) (PET)-based membrane having a hydrophilic surface, high nutrient ion permeability, sufficient mechanical strength, and organic fouling resistance, using an anthracene (ANT)-attached polyethylene glycol (PEG) surface modification agent (SMA) synthesized in this work. During the modification process, the ANT parts of the SMAs poke through and anchor to the surface of a commercial PET woven fabric via physical interactions and mechanical locking. The PEG chain parts coat the surface in the brush and arch forms, which generates a hydration layer on the fabric surface. The consequently obtained surface property and unique structure of the modified PET-based membrane result in higher nitrate ion permeability, organic fouling resistance, and microalgae production compared to those of the unmodified one. These are also affected by the molecular weight of the PEG and the number density of the anchored SMAs. The study demonstrates that this new surface modification method has the potential to allow the development of a desirable PET-based membrane for the efficient massive production of marine microalgae.
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Incrustaciones Biológicas/prevención & control , Chlorophyta/crecimiento & desarrollo , Técnicas de Cultivo/instrumentación , Membranas Artificiales , Microalgas/crecimiento & desarrollo , Tereftalatos Polietilenos/química , Antracenos/química , Reactores Biológicos/microbiología , Permeabilidad , Polietilenglicoles/química , Propiedades de SuperficieRESUMEN
PURPOSE: To compare bacteria recovered by standard cultures and metataxonomics, particularly with regard to ventilator-associated pneumonia (VAP) pathogens, and to determine if the presence of particular bacteria or microbiota in tracheal and oropharyngeal secretions during the course of intubation was associated with the development of VAP. METHODS: In this case-control study, oropharyngeal secretions and endotracheal aspirate were collected daily in mechanically ventilated patients. Culture and metataxonomics (16S rRNA gene-based taxonomic profiling of bacterial communities) were performed on serial upper respiratory samples from patients with late-onset definite VAP and their respective controls. RESULTS: Metataxonomic analyses showed that a low relative abundance of Bacilli at the time of intubation in the oropharyngeal secretions was strongly associated with the subsequent development of VAP. On the day of VAP, the quantity of human and bacterial DNA in both tracheal and oropharyngeal secretions was significantly higher in patients with VAP than in matched controls with similar ventilation times. Molecular techniques identified the pathogen(s) of VAP found by culture, but also many more bacteria, classically difficult to culture, such as Mycoplasma spp. and anaerobes. CONCLUSIONS: Molecular analyses of respiratory specimens identified markers associated with the development of VAP, as well as important differences in the taxa abundance between VAP and controls. Further prospective trials are needed to test the predictive value of these markers, as well as the relevance of uncultured bacteria in the pathogenesis of VAP.
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Biomarcadores/análisis , Microbiota , Neumonía Asociada al Ventilador/microbiología , APACHE , Adulto , Anciano , Estudios de Casos y Controles , Estudios de Cohortes , Técnicas de Cultivo/instrumentación , Técnicas de Cultivo/métodos , Femenino , Humanos , Unidades de Cuidados Intensivos/organización & administración , Masculino , Persona de Mediana Edad , Orofaringe/microbiología , Neumonía Asociada al Ventilador/mortalidad , Estudios Prospectivos , ARN Ribosómico 16S/análisis , Respiración Artificial/efectos adversos , Suiza , Tráquea/microbiologíaRESUMEN
The root system of plants is a major component of their bodies in terms of both function and bulk. The investigation of root system development is greatly assisted by microfluidic devices, which improve the spatial and temporal resolution of observations without destroying tissue. In the present study, a multi-chamber petaloid root-growth chip was developed for studying the development and physiology of root systems that have thin branching structures (i.e., fibrous root systems). The petaloid root-growth chip includes a central seed germination chamber and five root-growth chambers for observing the development of fibrous roots. The proposed device was applied for investigating the root system development of Oryza sativa. The phenotype and growth kinetics of O. sativa root systems grown in the proposed device were compared with those obtained during growth in a conventional conical flask with agar-based medium, and the results indicated that cultivation in the miniaturized device did not delay root system growth in the early stage (≤2 weeks). In addition, the transparent device enabled the non-destructive observation of the developmental and microstructural characteristics of the roots, such as the root caps, root border cells, and root hairs. Moreover, the ability to control the microenvironment in each of the five root-growth chambers individually facilitated the investigation of specific adaptations in the fibrous root growth of single O. sativa seedlings to different drought stresses. Accordingly, five polyethylene glycol (PEG)6000-induced drought stress conditions were established in the five root-growth chambers to investigate the root development of a single O. sativa seedling in the central germination chamber. In situ observations demonstrated that the different PEG6000-induced conditions affected the root growth responses and root microstructural adaptations of the single seedlings in each root-growth chamber. Therefore, the petaloid root-growth microfluidic chip can eliminate the effects of variations in different plant seeds to reveal the responses of plants to different environmental conditions more objectively while concurrently allowing for non-destructive observations at very high spatial and temporal resolution.
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Técnicas de Cultivo/instrumentación , Dispositivos Laboratorio en un Chip , Oryza/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , Sequías , Germinación , Oryza/fisiología , Raíces de Plantas/fisiología , Semillas/crecimiento & desarrollo , Estrés FisiológicoRESUMEN
Cells in the body collectively sustain mechanical deformations in almost all physiological functions. From the morphogenesis stage, cells' ability to sustain stress is essential for the body's well-being. Several pathologies have been associated with abnormal mechanical properties, thus suggesting the Young's modulus as a biomarker to diagnose diseases and determine their progression. Advancements in the field are quite slow because current techniques for measuring cell and tissue mechanics rely on complex and bulky measurement platforms that have low repeatability rates and limited measurement time-scales. We present the first miniaturized system that allows accurate quantification of the Young's modulus of adherent cell monolayers over a longer time (1-2 days). Our approach is based on tensile testing and optical read-out. Thanks to a thoughtful design and material choice, we are able to miniaturize tensile testing platforms into a 1 cm × 2 cm device. We provide highly repeatable Young's modulus measurements in the relevant range between 3 kPa and 300 kPa, over time and under physiological conditions, thus representing an interesting alternative to existing measurement platforms. Furthermore, the compatibility with standard biological equipment, continuous optical imaging and measurements on all types of adherent cells make this device highly versatile. Measurements on human sarcoma osteogenic (SaOS2) and Madin-Darby canine kidney cells (MDCK) are reported. The demonstrated capability to measure real-time changes in mechanical properties, such as after chemical treatment, opens the door for investigating the effects of drugs on cell mechanics.
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Técnicas de Cultivo , Elastómeros/química , Células de Riñón Canino Madin Darby/citología , Osteosarcoma/patología , Estrés Mecánico , Animales , Adhesión Celular , Línea Celular Tumoral , Técnicas de Cultivo/instrumentación , Perros , HumanosRESUMEN
Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. The objective of this study is to investigate the impacts of neural spheroids and vascular spheroids interactions on the regional brain-like tissue patterning in cortical spheroids derived from human iPSCs. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting human mesenchymal stem cells (MSCs). Single hybrid spheroids were constructed at different iNPC: iEC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in low-attachment 96-well plates. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-ß1 in hybrid spheroid system. In addition, tri-cultured spheroids had high levels of TBR1 (deep cortical layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and cell-cell communications on cortical spheroid and organoid patterning. Moreover, tri-culture system elevated blood-brain barrier gene expression (e.g., GLUT-1), CD31, and tight junction protein ZO1 expression. Treatment with AMD3100, a CXCR4 antagonist, showed the immobilization of MSCs during spheroid fusion, indicating a CXCR4-dependent manner of hMSC migration and homing. This forebrain-like model has potential applications in understanding heterotypic cell-cell interactions and novel drug screening in diseased human brain.
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Encéfalo , Técnicas de Cultivo , Organoides , Esferoides Celulares , Células Madre , Encéfalo/citología , Encéfalo/metabolismo , Proliferación Celular , Técnicas de Cultivo/instrumentación , Técnicas de Cultivo/métodos , Citocinas/metabolismo , Humanos , Neurogénesis/fisiología , Neuronas/citología , Neuronas/metabolismo , Organoides/citología , Organoides/metabolismo , Técnicas de Placa-Clamp , Esferoides Celulares/citología , Esferoides Celulares/metabolismo , Células Madre/citología , Andamios del TejidoRESUMEN
This paper reports a vision-based automated microfluidic system for morphological measurement and size-based sorting of the nematode worm C. elegans. Exceeding the capabilities of conventional worm sorting microfluidic devices purely relying on passive sorting mechanisms, our system is capable of accurate measurement of the worm length/width and active sorting of worms with the desired sizes from a mixture of worms with different body sizes. This function is realized based on the combination of real-time, vision-based worm detection and sizing algorithms and automated on-chip worm manipulation. A double-layer microfluidic device with computer-controlled pneumatic valves is developed for sequential loading, trapping, vision-based sizing, and sorting of single worms. To keep the system operation robust, vision-based algorithms on detecting multi-worm loading and worm sizing failure have also been developed. We conducted sorting experiments on 319 worms and achieved an average sorting speed of 10.4 worms per minute (5.8 s/worm) with an operation success rate of 90.3%. This system will facilitate the worm biology studies where body size measurement and size-based sorting of many worms are needed.
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Caenorhabditis elegans/aislamiento & purificación , Caenorhabditis elegans/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Algoritmos , Animales , Técnicas de Cultivo/instrumentación , Diseño de EquipoRESUMEN
Optimized culture conditions are essential for the investigation of biological processes. In this work, on-chip optimization of bacterial culture conditions by combining microfluidics with the Box-Behnken design response surface methodology is presented. With this methodology, the effects of several cultivation variables and their interactions were investigated enabling very fast drug susceptibility screening. The proposed measurement protocol for the determination of minimum inhibitory concentration (MIC) consist of three steps: i) single factor experiments to determine the effect of pH, nutrient concentration, and temperature on the bacterial culture; ii) analyses of the relationship between variables and the effect of the individual variables by means of the Box-Behnken design and response surface methodology (BBD-RSM) optimization; and iii) bacterial susceptibility screening of drugs and drug combinations. BBD-RSM is efficient to determine the optimal growth conditions of bacteria species with a strongly reduced amount of required experiments. On top of that, these experiments can in principle all be performed at the same time, yielding significant time-savings. The found optimized culture conditions of E. coli were applied to determine the MIC values of the drugs penicillin-streptomycin and baicalein, and combinations of those. MIC values were obtained within 8-14â¯h, including the 6-8â¯h required to determine the optimal growth parameters. The microfluidic BBD-RSM method results in a significant time reduction compared to the standard 2-4 days required to determine MIC values and is, therefore, a potential alternative in the management of bacterial infections.
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Antibacterianos/farmacología , Técnicas de Cultivo/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Dispositivos Laboratorio en un Chip , Pruebas de Sensibilidad Microbiana , Factores de TiempoRESUMEN
Despite low sensitivity, culture of periprosthetic tissue (PPT) specimens on agars and in broths has traditionally been used for the detection of causative microorganisms in patients suspected for prosthetic joint infection (PJI). The aim of this study was to evaluate the added diagnostic value of culturing PPT in blood culture bottles (BCB) over the conventional combination of standard agar and broth alone. This prospective cohort study was conducted over a 12-month period and included consecutive patients undergoing revision arthroplasty. Overall, 113 episodes from 90 subjects were studied; 45 subjects (50.0%) met the Infectious Diseases Society of America (IDSA) criteria for PJI, of whom the majority (75.6%) had an acute infection. Sensitivity and specificity of culture were assessed using IDSA criteria for PJI as gold standard. Although the increase in sensitivity from 84.44 (CI 70.54; 93.51) to 93.33% (81.73; 98.60) was not significant, added diagnostic value of culturing PPT in BCBs was demonstrated by the significantly higher number of detected pathogens in culture sets with BCBs compared to culture without BCBs (61 pathogens in conventional set versus 89 when BCBs were included for 57 PJI episodes, P = <0.0001). In 17 (29.8%) episodes, microorganisms were cultured from BCBs only, and in 9 (52.9%) of these episodes, virulent pathogens were found. This study demonstrates that PPT culture in BCBs leads to isolation of additional microorganisms, both virulent and low-virulent, which were not cultured with use of agars and broths alone. Isolation of additional causative microorganisms has serious consequences for the treatment strategy in PJI.
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Bacterias/aislamiento & purificación , Técnicas de Cultivo/métodos , Prótesis Articulares/microbiología , Técnicas Microbiológicas/métodos , Infecciones Relacionadas con Prótesis/diagnóstico , Infecciones Relacionadas con Prótesis/patología , Anciano , Anciano de 80 o más Años , Artroplastia , Técnicas de Cultivo/instrumentación , Humanos , Prótesis Articulares/efectos adversos , Estudios Prospectivos , Infecciones Relacionadas con Prótesis/cirugía , Reoperación , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The nematode Caenorhabditis elegans is increasingly used as a model for human biology. However, in vivo culturing platforms for C. elegans allowing high-content phenotyping during their life cycle in an automated fashion are lacking so far. Here, a multiplexed microfluidic platform for the rapid high-content phenotyping of populations of C. elegans down to single animal resolution is presented. Nematodes are (i) reversibly and regularly confined during their life inside tapered channels for imaging fluorescence signal expression and to measure their growth parameters, and (ii) allowed to freely move in microfluidic chambers, during which the swimming behavior was video-recorded. The obtained data sets are analyzed in an automated way and 19 phenotypic parameters are extracted. Our platform is employed for studying the effect of bacteria dilution, a form of dietary restriction (DR) in nematodes, on a worm model of Huntington's disease and demonstrates the influence of DR on disease regression.
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Caenorhabditis elegans/fisiología , Larva/fisiología , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Animales , Técnicas de Cultivo/instrumentación , Técnicas de Cultivo/métodos , Modelos Animales de Enfermedad , Diseño de Equipo , FenotipoRESUMEN
I have reared a culture of the tardigrade Hypsibius exemplaris for 30 years, since 1987. Here, I present my culture protocol.
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Chlorophyta/crecimiento & desarrollo , Técnicas de Cultivo/métodos , Tardigrada/crecimiento & desarrollo , Animales , Técnicas de Cultivo/instrumentación , Factores de TiempoRESUMEN
Many species of tardigrades can survive severe water loss, but different species tolerate different desiccation conditions. Hypsibius exemplaris is able to survive desiccation after an initial period of slow drying, as described here. This protocol will likely work for other tardigrade species as well. Drying of tardigrades can be used for probing the mechanistic underpinnings of desiccation tolerance, as well as for practical purposes such as shipping and long-term storage of the animals.
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Adaptación Fisiológica/fisiología , Desecación/instrumentación , Desecación/métodos , Tardigrada/fisiología , Animales , Técnicas de Cultivo/instrumentación , Técnicas de Cultivo/métodos , HumedadRESUMEN
Aquatic organisms are impacted by various biotic and abiotic stressors such as current, inter- and intraspecific competition for food resources and habitat, neobiota as well as an increasing number of chemicals. The latter also include pharmaceuticals, which are increasingly being detected in surface waters due to their growing use. The aim of our study was to determine effect data for metoprolol as a model compound for beta-blockers under an environmentally realistic exposure scenario on aquatic invertebrates inhabiting lotic environments. To this end we performed a 40-day experiment in artificial indoor streams (AIS) located in a greenhouse. We focussed on three autochthonous invertebrate species with high relevance in stream ecology: the amphipod Gammarus fossarum, the gastropod Potamopyrgus antipodarum, and the oligochaete Lumbriculus variegatus. Effects on reproduction were found with EC10 (40 days) values of 0.092 mg L-1 (G. fossarum), 0.253 mg L-1 (P. antipodarum), and 0.596 mg L-1 (L. variegatus). Considering environmental data, metoprolol seems to pose no hazard for aquatic invertebrates at present exposure levels.
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Organismos Acuáticos/efectos de los fármacos , Ecosistema , Ambiente Controlado , Invertebrados/efectos de los fármacos , Metoprolol/farmacología , Anfípodos/efectos de los fármacos , Anfípodos/crecimiento & desarrollo , Animales , Organismos Acuáticos/crecimiento & desarrollo , Técnicas de Cultivo/instrumentación , Técnicas de Cultivo/métodos , Gastrópodos/efectos de los fármacos , Gastrópodos/crecimiento & desarrollo , Invertebrados/crecimiento & desarrollo , Invertebrados/fisiología , Oligoquetos/efectos de los fármacos , Oligoquetos/crecimiento & desarrollo , Reproducción/efectos de los fármacos , Ríos , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/farmacologíaRESUMEN
Continuous culture techniques were developed in the early twentieth century to replace cumbersome studies of cell growth in batch cultures. In contrast to batch cultures, they constituted an open concept, as cells are forced to proliferate by adding new medium while cell suspension is constantly removed. During the 1940s and 1950s new devices have been designed-called "automatic syringe mechanism," "turbidostat," "chemostat," "bactogen," and "microbial auxanometer"-which allowed increasingly accurate quantitative measurements of bacterial growth. With these devices cell growth came under the external control of the experimenters and thus accessible for developing a mathematical theory of growth kinetics-developed mainly by Jacques Monod, Aron Novick and Leo Szilard in the early 1950s and still in use today. The paper explores the development of continuous culture devices and claims that these devices are simulators for standard cells following specific requirements, in particular involving mathematical constraints in the design and setting of the devices as well as experiments. These requirements have led to contemporary designs of continuous culture techniques realizing a specific event-based flow algorithm able to simulate directed evolution and produce artificial cells and microorganisms. This current development is seen as an alternative approach to today's synthetic biology.
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Técnicas de Cultivo/historia , Microbiología/historia , Técnicas de Cultivo/instrumentación , Historia del Siglo XX , Microbiología/instrumentación , Proyectos de InvestigaciónRESUMEN
Detection of pathogenic bacteria and monitoring their susceptibility to antibiotics are of great importance in the fields of medicine, pharmaceutical research, as well as water and food industries. In order to develop a photonic biosensor for detection of bacteria by taking advantage of photoluminescence (PL) of GaAs-based devices, we have investigated the capture and growth of Escherichia coli K12 on bare and biofunctionalized surfaces of GaAs (001) - a material of interest for capping different semiconductor microstructures. The results were compared with the capture and growth of Escherichia coli K12 on Au surfaces that have commonly been applied for studying a variety of biological and biochemical reactions. We found that neither GaAs nor Au-coated glass wafers placed in Petri dishes inoculated with bacteria inhibited bacterial growth in nutrient agar, regardless of the wafers being bare or biofunctionalized. However, the capture and growth of bacteria on biofunctionalized surfaces of GaAs and Au wafers kept in a flow cell and exposed to different concentrations of bacteria and growth medium revealed that the initial surface coverage and the subsequent bacterial growth were dependent on the biofunctionalization architecture, with antibody-coated surfaces clearly being most efficient in capturing bacteria and offering better conditions for growth of bacteria. We have observed that, as long as the GaAs wafers were exposed to bacterial suspensions at concentrations of at least 105 CFU/mL, bacteria could grow on the surface of wafers, regardless of the type of biofunctionalization architecture used to capture the bacteria. These results provide important insight towards the successful development of GaAs-based devices designed for photonic monitoring of bacterial reactions to different biochemical environments.
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Arsenicales/farmacología , Técnicas de Cultivo/métodos , Escherichia coli K12/efectos de los fármacos , Escherichia coli K12/crecimiento & desarrollo , Galio/farmacología , Arsenicales/química , Técnicas de Cultivo/instrumentación , Galio/química , Oro/química , Propiedades de SuperficieRESUMEN
We have miniaturized standard culture techniques to rear arrays of isolated, individual C. elegans throughout their lives on solid gel media. The resulting apparatus is compatible with brightfield and fluorescence microscopy, enabling longitudinal studies of morphology and fluorescent transgene expression. Our culture system exploits a novel crosslinking reaction between a polyethylene glycol hydrogel and a silicone elastomer to constrain animals to individual "corrals" on the gel surface. These devices are simple to construct on the benchtop with commercially available reagents, and, unlike microfluidic isolation methods, do not rely on micropatterned materials. We demonstrate that this new culture method has negligible effects on the physiology of C. elegans compared to standard culture on agar plates. In addition, RNAi techniques are effective in this system. Finally, the hydrogel-silicone binding chemistry that we developed also allows traditional microfluidic devices to be covalently attached to gel substrates instead of glass.
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Caenorhabditis elegans/fisiología , Técnicas de Cultivo/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Microscopía Fluorescente/instrumentación , Animales , Técnicas de Cultivo/métodos , Diseño de Equipo , Hidrogeles , Dispositivos Laboratorio en un Chip , Microscopía Fluorescente/métodos , PolietilenglicolesRESUMEN
In the paper, the microaquarium fabricated in a form of entirely glass lab-on-a-chip for culturing and microscale study of microorganisms has been presented. A new approach towards cellular studies that brings a significant improvement over commonly utilized - polymer-based solutions has been shown. For the first time, all-borosilicate glass chip was applied for the culturing of the selected microorganisms and enabled notable population growth and behaviorism investigation. The chip fabrication method in comparison to typical glass chip technology was notably simplified, including quick patterning and low temperature bonding in 80 °C. In the studies, both a single-cell (Euglena gracilis and Euglena viridis) and multi-cell microorganisms (Lepadella patella) were cultured in the microaquarium. Behaviorism of the selected microorganisms was investigated by supplying various proportions of carbon dioxide, nitrogen and air into the chip. Tests included studies of microorganisms chemotaxis, viability (mostly based on photosynthesis process) and coexistence in the lab-on-a-chip environment. The experiments confirmed that the developed chip is a tool that fits the requirements for the culturing and behavioral studies of microorganisms and constitute ground-works to propel its further application in broadly defined cellular study field.
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Técnicas de Cultivo/instrumentación , Euglena gracilis/crecimiento & desarrollo , Vidrio , Dispositivos Laboratorio en un Chip , Rotíferos/crecimiento & desarrollo , Animales , Quimiotaxis , Euglena gracilis/citología , Euglena gracilis/metabolismo , Fotosíntesis , Rotíferos/citología , Rotíferos/metabolismoRESUMEN
Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.
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Productos Biológicos/farmacología , Técnicas de Cultivo/métodos , Metano/metabolismo , Methanobrevibacter/efectos de los fármacos , Methanococcus/efectos de los fármacos , Rumen/microbiología , Rumiantes/microbiología , Animales , Técnicas de Cultivo/instrumentación , Evaluación Preclínica de Medicamentos , Methanobrevibacter/crecimiento & desarrollo , Methanobrevibacter/metabolismo , Methanococcus/crecimiento & desarrollo , Methanococcus/metabolismo , Rumen/metabolismo , Rumiantes/metabolismoRESUMEN
Laboratory investigations of physiological processes in phytoplankton require precise control of experimental conditions. Chemostats customized to control and maintain stable pH levels (pHstats) are ideally suited for investigations of the effects of pH on phytoplankton physiology, for example in context of ocean acidification. Here we designed and constructed a simple, flexible pHstat system and demonstrated its operational capabilities under laboratory culture conditions. In particular, the system is useful for simulating natural cyclic pH variability within aquatic ecosystems, such as diel fluctuations that result from metabolic activity or tidal mixing in estuaries. The pHstat system operates in two modes: (1) static/set point pH, which maintains pH at a constant level, or (2) dynamic pH, which generates regular, sinusoidal pH fluctuations by systematically varying pH according to user-defined parameters. The pHstat is self-regulating through the use of interchangeable electronically controlled reagent or gas-mediated pH-modification manifolds, both of which feature flow regulation by solenoid valves. Although effective pH control was achieved using both liquid reagent additions and gas-mediated methods, the liquid manifold exhibited tighter control (±0.03pH units) of the desired pH than the gas manifold (±0.10pH units). The precise control provided by this pHstat system, as well as its operational flexibility will facilitate studies that examine responses by marine microbiota to fluctuations in pH in aquatic ecosystems.