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Frontiers in Microbiology 2022In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is...
In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is essential. In this study, we fabricated a gel-filled microwell array device using resin for microbial culture. The device has an integrated sealing mechanism that enables high-density isolation based on the culture of microorganisms; the device is easily manageable, facilitating observation using bright-field microscopy. This low-cost device made from polymethyl methacrylate (PMMA)/polyethylene terephthalate (PET) has 900 microwells (600 μm × 600 μm × 700 μm) filled with a microbial culture gel medium in glass slide-sized plates. It also has grooves for maintaining the moisture content in the micro-gel. The partition wall between the wells has a highly hydrophobic coating to inhibit microbial migration to neighboring wells and to prevent exchange of liquid substances. After being hermetically sealed, the device can maintain moisture in the agarose gels for 7 days. In the bacterial culture experiment using this device, environmental bacteria were isolated and cultured in individual wells after 3 days. Moreover, the isolated bacteria were then picked up from wells and re-cultured. This device is effective for the first screening of microorganisms from marine environmental samples.
PubMed: 36590440
DOI: 10.3389/fmicb.2022.1031439 -
Molecular Biology and Evolution Jan 2014In the 1960s-1980s, determination of bacterial growth rates was an important tool in microbial genetics, biochemistry, molecular biology, and microbial physiology. The...
In the 1960s-1980s, determination of bacterial growth rates was an important tool in microbial genetics, biochemistry, molecular biology, and microbial physiology. The exciting technical developments of the 1990s and the 2000s eclipsed that tool; as a result, many investigators today lack experience with growth rate measurements. Recently, investigators in a number of areas have started to use measurements of bacterial growth rates for a variety of purposes. Those measurements have been greatly facilitated by the availability of microwell plate readers that permit the simultaneous measurements on up to 384 different cultures. Only the exponential (logarithmic) portions of the resulting growth curves are useful for determining growth rates, and manual determination of that portion and calculation of growth rates can be tedious for high-throughput purposes. Here, we introduce the program GrowthRates that uses plate reader output files to automatically determine the exponential portion of the curve and to automatically calculate the growth rate, the maximum culture density, and the duration of the growth lag phase. GrowthRates is freely available for Macintosh, Windows, and Linux. We discuss the effects of culture volume, the classical bacterial growth curve, and the differences between determinations in rich media and minimal (mineral salts) media. This protocol covers calibration of the plate reader, growth of culture inocula for both rich and minimal media, and experimental setup. As a guide to reliability, we report typical day-to-day variation in growth rates and variation within experiments with respect to position of wells within the plates.
Topics: Algorithms; Bacteria; Bacteriological Techniques; Culture Media; Phenotype; Reproducibility of Results; Software
PubMed: 24170494
DOI: 10.1093/molbev/mst187 -
The Journal of Reproduction and... Aug 2010We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for...
We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.
Topics: Animals; Cattle; Cell Count; Cells, Cultured; Dimethylpolysiloxanes; Embryo Culture Techniques; Embryo, Mammalian; Embryonic Development; Fertilization in Vitro; Microchemistry; Models, Biological; Parthenogenesis
PubMed: 20484872
DOI: 10.1262/jrd.09-213h -
Journal of Clinical Microbiology Mar 1995We developed a colorimetric microwell plate hybridization assay (CoMPHA) for the specific detection of 5'-biotinylated amplified Mycobacterium tuberculosis DNA. The... (Comparative Study)
Comparative Study
We developed a colorimetric microwell plate hybridization assay (CoMPHA) for the specific detection of 5'-biotinylated amplified Mycobacterium tuberculosis DNA. The optical densities of the CoMPHA corresponded to the initial amounts of purified template DNA. Here, we show that the CoMPHA is useful in distinguishing the PCR-positive and PCR-negative samples.
Topics: Base Sequence; Colorimetry; DNA Probes; DNA, Viral; Humans; Molecular Sequence Data; Mycobacterium tuberculosis; Nucleic Acid Hybridization; Polymerase Chain Reaction; Sputum
PubMed: 7751391
DOI: 10.1128/jcm.33.3.752-754.1995 -
Medicina (Kaunas, Lithuania) Feb 2023This study presents the development and validation of the 96-microwell-based spectrofluorimetric (MW-SFL) and high performance liquid chromatography (HPLC) with...
Development of Novel Microwell-Based Spectrofluorimetry and High-Performance Liquid Chromatography with Fluorescence Detection Methods and High Throughput for Quantitation of Alectinib in Bulk Powder and Urine Samples.
This study presents the development and validation of the 96-microwell-based spectrofluorimetric (MW-SFL) and high performance liquid chromatography (HPLC) with fluorescence detection (HPLC-FD) methods for the quantitation of alectinib (ALC) in its bulk powder form and in urine samples. The MW-SFL was based on the enhancement of the native fluorescence of ALC by the formation of micelles with the surfactant cremophor RH 40 (Cr RH 40) in aqueous media. The MW-SFL was executed in a 96-microwell plate and the relative fluorescence intensity (RFI) was recorded by utilizing a fluorescence plate reader at 450 nm after excitation at 280 nm. The HPLC-FD involved the chromatographic separation of ALC and ponatinib (PTB), as an internal standard (IS), on a C column and a mobile phase composed of methanol:potassium dihydrogen phosphate pH 7 (80:20, /) at a flow rate of 2 mL min. The eluted ALC and PTB were detected by utilizing a fluorescence detector set at 365 nm for excitation and 450 nm for emission. Validation of the MW-SFL and HPLC-FD analytical methods was carried out in accordance with the recommendations issued by the International Council for Harmonization (ICH) for the process of validating analytical procedures. Both methods were efficaciously applied for ALC quantitation in its bulk form as well as in spiked urine; the mean recovery values were ≥86.90 and 95.45% for the MW-SFL and HPLC-FD methods, respectively. Both methodologies are valuable for routine use in quality control (QC) laboratories for determination of ALC in pure powder form and in human urine samples.
Topics: Humans; Chromatography, High Pressure Liquid; Powders; Piperidines; Carbazoles
PubMed: 36984441
DOI: 10.3390/medicina59030441 -
RSC Advances May 2024Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene...
Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene (PS) microwell plates form a part of this waste. These plates are the backbone of high throughput colorimetric measurements in academic, research, and healthcare settings for detection/quantification of wide-ranging analytes including proteins, carbohydrates, nucleic acids, and enzyme activity. Polystyrene (PS) microwell plates serve as a platform for holding samples and reagents, where mixing initiates chemical reaction(s), and the ensuing color changes are quantified using a microplate reader. However, these plates are rarely reused or recycled, contributing to the staggering amounts of plastic waste generated in scientific laboratories. Here, we are reporting the fabrication of cellulose acetate (CA) microwell plates as a greener alternative to non-biodegradable PS plates and we demonstrate their application in colorimetric assays. These easy to fabricate, lighter weight, customizable, and environmentally friendly plates were fabricated in 96- and 384-well formats and made water impermeable through chemical treatment. The plates were tested in three different colorimetric analyses: (i) bicinchoninic acid assay (BCA) for protein quantification; (ii) chymotrypsin (CT) activity assay; and (iii) alkaline phosphatase (AP) activity assay. Color intensities were quantified using a freely available smartphone application, Spotxel® Reader (Sicasys Software GmbH). To benchmark the performance of this platform, the same assays were performed in commercial PS plates too and quantified using a UV/Vis microplate reader. The two systems yielded comparable linear correlation coefficients, LOD and LOQ values, thereby validating the CA plate-cell phone based analytical method. The CA microwell plates, coupled with smart phone optical data capture, provide greener, accessible, and scalable tools for all laboratory settings and are particularly well-suited for resource- and infrastructure-limited environments.
PubMed: 38741966
DOI: 10.1039/d4ra01317d -
Analytical Chemistry Jun 2001A new design for high-throughput microfabricated capillary electrophoresis/electrospray mass spectrometry (CE/ ESI-MS) with automated sampling from a microwell plate is...
A new design for high-throughput microfabricated capillary electrophoresis/electrospray mass spectrometry (CE/ ESI-MS) with automated sampling from a microwell plate is presented. The approach combines a sample-loading port, a separation channel, and a liquid junction, the latter for coupling the device to the MS with a miniaturized subatmospheric electrospray interface. The microdevice was attached to a polycarbonate manifold with external electrode reservoirs equipped for electrokinetic and pressure-fluid control. A computer-activated electropneumatic distributor was used for both sample loading from the microwell plate and washing of channels after each run. Removal of the electrodes and sample reservoirs from the microdevice structure significantly simplified the chip design and eliminated the need both for drilling access holes and for sample/buffer reservoirs. The external manifold also allowed the use of relatively large reservoirs that are necessary for extended time operation of the system. Initial results using this microfabricated system for the automated CE/ESI-MS analysis of peptides and protein digests are presented.
Topics: Amino Acid Sequence; Electrophoresis, Capillary; Molecular Sequence Data; Peptides; Proteins; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Trypsin
PubMed: 11403316
DOI: 10.1021/ac001432v -
Clinical Infectious Diseases : An... Apr 1995Infections of the CNS with the nonpolio enteroviruses are common and important causes of morbidity in both children and adults. Studies have recently defined the... (Review)
Review
Infections of the CNS with the nonpolio enteroviruses are common and important causes of morbidity in both children and adults. Studies have recently defined the short-term and long-term outcomes of aseptic meningitis due to the enteroviruses. Focal encephalitis is increasingly recognized as a complication of enterovirus infection. Patients at greatest risk for sequelae of CNS enteroviral disease include neonates and those who are immunocompromised. The clinical presentation may mimic that of bacterial or other viral CNS infections, a circumstance making laboratory diagnosis of paramount importance for reducing unnecessary hospitalization and therapy. Recent advances in PCR technology, including its adaptation to a colorimetric microwell plate format, promise to greatly facilitate diagnosis of enteroviral infections. Promising antiviral drugs for CNS disease and other serious manifestations of enteroviral infections are under development.
Topics: Adult; Central Nervous System Diseases; Child; DNA, Viral; Encephalitis; Enterovirus; Enterovirus Infections; Humans; Infant, Newborn; Meningitis; Polymerase Chain Reaction
PubMed: 7795102
DOI: 10.1093/clinids/20.4.971 -
Communications Biology Nov 2023The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent...
The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.
Topics: Optogenetics; Feedback; Escherichia coli; Algorithms; Spectrum Analysis
PubMed: 38001175
DOI: 10.1038/s42003-023-05532-4 -
Applied and Environmental Microbiology May 2022The capacity to defluorinate polyfluorinated organic compounds is a rare phenotype in microbes but is increasingly considered important for maintaining the environment....
The capacity to defluorinate polyfluorinated organic compounds is a rare phenotype in microbes but is increasingly considered important for maintaining the environment. New discoveries will be greatly facilitated by the ability to screen many natural and engineered microbes in a combinatorial manner against large numbers of fluorinated compounds simultaneously. Here, we describe a low-volume, high-throughput screening method to determine defluorination capacity of microbes and their enzymes. The method is based on selective binding of fluoride to a lanthanum chelate complex that gives a purple-colored product. It was miniaturized to determine biodefluorination in 96-well microtiter plates by visual inspection or robotic handling and spectrophotometry. Chemicals commonly used in microbiological studies were examined to define usable buffers and reagents. Base-catalyzed, purified enzyme and whole-cell defluorination reactions were demonstrated with fluoroatrazine and showed correspondence between the microtiter assay and a fluoride electrode. For discovering new defluorination reactions and mechanisms, a chemical library of 63 fluorinated compounds was screened with Pseudomonas putida F1 in microtiter well plates. These data were also calibrated against a fluoride electrode. Our new method revealed 21 new compounds undergoing defluorination. A compound with four fluorine substituents, 4-fluorobenzotrifluoride, was shown to undergo defluorination to the greatest extent. The mechanism of its defluorination was studied to reveal a latent microbial propensity to defluorinate trifluoromethylphenyl groups, a moiety that is commonly incorporated into numerous pharmaceutical and agricultural chemicals. Thousands of organofluorine chemicals are known, and a number are considered to be persistent and toxic environmental pollutants. Environmental bioremediation methods are avidly being sought, but few bacteria biodegrade fluorinated chemicals. To find new organofluoride biodegradation, a rapid screening method was developed. The method is versatile, monitoring chemical, enzymatic, and whole-cell biodegradation. Biodegradation of organofluorine compounds invariably releases fluoride anions, which was sensitively detected. Our method uncovered 21 new microbial defluorination reactions. A general mechanism was delineated for the biodegradation of trifluoromethylphenyl groups that are increasingly being used in drugs and pesticides.
Topics: Biodegradation, Environmental; Fluorides; Fluorine; Pseudomonas putida
PubMed: 35435713
DOI: 10.1128/aem.00288-22