22 research outputs found

    Robust estimation of bacterial cell count from optical density

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    Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

    Refinement of a radioreceptor binding assay for nicotinic acid adenine dinucleotide phosphate

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    The measurement of changes in nicotinic acid adenine dinucleotide phosphate (NAADP) levels in cells has been, and remains, key to the investigation of the functions of NAADP as a Ca2+-releasing second messenger. Here we provide details of how to isolate NAADP from cells by extraction with perchloric acid and then measure the NAADP using a radioreceptor assay. We demonstrate that NAADP is neither generated nor broken down during sample processing conditions and that radioreceptor assay is highly selective for the detection of NAADP under cell extract conditions. Furthermore, a number of improvements, such as solid-state detection of the radioactivity, are incorporated to enhance the safety of the procedure. Finally, we have developed a new method to prevent the endogenous metabolism of NAADP by chelating Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), thereby reducing the difficulty of catching a small transient rise in NAADP levels. In summary, we have refined and improved a method for measuring NAADP levels and presented it in a manner accessible to a wide range of laboratories. It is expected that this will enhance research in the NAADP field
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