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Counting bacteria in microfluidic devices: smartphone compatible ‘dip-and-test’ viable cell quantitation using resazurin amplified detection in microliter capillary arrays
Full text linkViable bacterial cell counting is fundamental to analytical microbiology and agar plate colony counting remains common yet laborious and slow. Here, we demonstrate two methods for counting bacteria using commercially available microfluidic devices. We show that accurate viable cell counting is possible using simple and easy ‘dip and test’ arrays of microcapillaries. Colorimetric and fluorescent growth detection both permit viable cell counting in microcapillaries either by limiting dilution into multiple microfluidic compartments using a single endpoint measurement, or alternatively by quantifying growth kinetics. The microcapillary devices are compatible with conventional 96 well plates and multichannel pipettes, expanding each microplate row into 120 individual 1 or 2 microlitre samples. At limiting dilution, counting the proportion of positive compartments permitted accurate calculation of gram-negative and gram-positive bacteria (E. coli and S. saprophyticus) at concentrations down to as low as 10 CFU/mL with almost 1:1 agreement with agar plate colony counts over four orders of magnitude. A smartphone camera was sufficient to record endpoint images of resazurin growth detection both colorimetrically and fluorescently. Viable cell counting of E. coli and S. saprophyticus was also possible through recording growth kinetics and determining the time taken to detect resazurin conversion. However, only the limiting dilution method remained consistent in the presence of urine matrix, as some interference in growth rate was observed when bacteria were spiked into higher concentrations of normal urine to simulate urinary tract infection patient samples. However, with the limiting dilution counting method endpoint growth was always detected even in the presence of 90% urine matrix, suggesting that this method might permit bacterial pathogen counting directly in clinical samples without agar plating
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Direct microfluidic antibiotic resistance testing in urine with smartphone capture: significant variation in sample matrix interference between individual human urine samples
Full text linkRapid and portable direct tests for antibiotic resistance in human clinical samples such as urine could reduce misuse of precious antimicrobials, by allowing treatment decisions to be informed by microfluidic diagnostic tests. We demonstrate that the variable composition of human urine can significantly affect the antibiotic minimum inhibitory concentration (MIC) measured using microfluidic devices. The urine sample matrix interference was not observed in pooled normal urine, emphasising the critical importance of assessing matrix interference with a wide range of individual urine samples, rather than a few standardised or pooled controls. Both dilution into assay medium and inclusion of buffer could reduce the matrix interference, but dilution may affect analytical sensitivity by increasing the minimum bacterial cell density needed in a sample for growth to be detected, especially for miniaturised devices that test small sample volumes. We conclude it is vital to fully assess and optimise novel analytical microbiology tools using multiple individual urine samples, otherwise the high variation in matrix interference will compromise the clinical performance of these rapid diagnostics that are urgently needed to tackle the global threat of antimicrobial resistance
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Challenges in microfluidic and point-of-care phenotypic antimicrobial resistance tests
Full text linkTo combat the threat to public health of antimicrobial resistance, there is a need for faster, more portable diagnostic tools to aid in antibiotic selection. Current methods for determining antimicrobial resistance of pathogens in clinical samples take days to result and require high levels of user input. Microfluidics offers many potential benefits, reducing time to result, user input, and allowing point of care testing. This review focuses on the challenges of developing functional or phenotypic microfluidic antimicrobial susceptibility tests; such methods complement other vital tools such as nucleic acid detection. Some of the most important challenges identified here are not unique to microfluidics but apply to most antimicrobial susceptibility testing innovations and relate to the nature of the sample being tested. For many high priority samples, mixtures of bacteria, highly variable target cell density, and the sample matrix can all affect measurements, and miniaturization can create sensitivity problems if target bacteria are dilute. Recent advances including smartphone capability, new sensors, microscopy, and a resurgence in paper microfluidics offer important opportunities for microfluidic engineering to simplify functional and phenotypic antimicrobial susceptibility testing. But the complexity of most clinical samples remains one of the biggest barriers to rapid uptake of microfluidics for antimicrobial resistance testing
Dilution Reduces Sample Matrix Effects for Rapid, Direct, and Miniaturised Phenotypic Antibiotic Susceptibility Tests for Bovine Mastitis
Full text linkThe 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
PiRamid: a compact Raspberry Pi imaging box to automate small-scale time-lapse digital analysis, suitable for laboratory and field use
No full textDigital imaging permits the quantitation of many experiments, such as microbiological growth assays, but laboratory digital imaging systems can be expensive and too specialised. The Raspberry Pi camera platform makes automated, controlled imaging affordable with accessible customisation. When combined with open source software and open-source 3D printed hardware, the control over image quality and capture of this platform permits the rapid development of novel instrumentation. Here we present “PiRamid”, a compact, portable, and inexpensive enclosure for autonomous imaging both in the laboratory and in the field. The modular three-piece 3D printed design makes it easy to incorporate different camera systems or lighting configurations (e.g., single wavelength LED for fluorescence). The enclosed design allows complete control of illumination unlike a conventional digital camera or smartphone, on a tripod or handheld, under ambient lighting. The stackable design permits rapid sample addition or camera focus adjustment, with a corresponding change in magnification and resolution. The entire unit is small enough to fit within a microbiological incubator, and cheap enough (∼£100) to scale out for larger parallel experiments. Simply, Python scripts fully automate illumination and image capture for small-scale experiments with an ∼110×85 mm area at 70–90 µm resolution. We demonstrate the versatility of PiRamid by capturing time-resolved, quantitative image data for a wide range of assays. Bacterial growth kinetics was captured for conventional microbiology (agar Petri dishes), 3D printed custom microbiology labware and microfluidic microbiology. To illustrate application beyond microbiology, we demonstrate time-lapse imaging of crystal growth and degradation of salad leaves. Minor modifications permit epi-illumination by addition of a LED ring to the camera module. We conclude that PiRamid permits inexpensive digital capture and quantitation of a wide range of experiments by time-lapse imaging to simplify both laboratory and field imaging
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Remote videolink observation of model home sampling and home testing devices to simplify usability studies for point-of-care diagnostics
Full text linkBoth home sample collection and home testing using rapid point-of-care diagnostic devices can offer benefits over attending a clinic/hospital to be tested by a healthcare professional. Usability is critical to ensure that in-home sampling or testing by untrained users does not compromise analytical performance. Usability studies can be laborious and rely on participants attending a research location or a researcher visiting homes; neither has been appropriate during COVID-19 outbreak control restrictions. We therefore developed a remote research usability methodology using videolink observation of home users. This avoids infection risks from home visits and ensures the participant follows the test protocol in their home environment. In this feasibility study, volunteers were provided with models of home blood testing and home blood sampling kits including a model lancet, sampling devices for dried blood spot collection, and model lateral flow device. After refining the study protocol through an initial pilot (n = 7), we compared instructions provided either as written instructions (n = 5), vs addition of video instructions (n = 5), vs written and video instructions plus videolink supervision by the researcher (n = 5). All users were observed via video call to define which test elements could be assessed remotely. All 22 participants in the study accessed the video call and configured their videolink allowing the researcher to clearly observe all testing tasks. The video call allowed the researcher to assess distinct errors during use including quantitative (volume of blood) and qualitative (inaccurate interpretation of results) errors many of which could compromise test accuracy. All participants completed the tasks and returned images of their completed tests (22/22) and most returned completed questionnaires (20/22). We suggest this remote observation via videolink methodology is a simple, rapid and powerful methodology to assess and optimise usability of point-of-care testing methods in the home setting
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Open Hardware for Microfluidics: Exploiting Raspberry Pi Singleboard Computer and Camera Systems for Customisable Laboratory Instrumentation
Full text linkThe integration of Raspberry Pi miniature computer systems with microfluidics has revolu-tionized the development of low-cost and customizable analytical systems in life science labor-atories. This review explores the applications of Raspberry Pi in microfluidics, with a focus on imaging, including microscopy and automated image capture. By leveraging the low-cost, flexi-bility and accessibility of Raspberry Pi components, high-resolution imaging and analysis have been achieved in direct mammalian and bacterial cellular imaging and a plethora of image based biochemical and molecular assays, from immunoassays, through microbial growth, to nucleic acid methods such as real-time-qPCR. The control of image capture permitted by Raspberry Pi hard-ware can also be combined with onboard image analysis. Open-source hardware offers an op-portunity to develop complex laboratory instrumentation systems at a fraction of the cost of commercial equipment and importantly, offer an opportunity to completely customise to meet the users’ needs. However, these benefits come with a trade-off: challenges remain for those wishing to incorporate open-source hardware equipment in their own work, including requirements for construction and operator skill, need for good documentation and the availability of rapid pro-totyping such as 3D printing plus other components. These advances in open-source hardware have the potential to improve efficiency, accessibility, and cost-effectiveness of microfluidic-based experiments and applications
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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