Robust estimation of bacterial cell count from optical density

Jacob Beal,Markus Gershater,Vinoo Selvarajah,Geoff Baldwin ,Russell Buckley-Taylor,John P Marken ,Abigail Sison,Traci Haddock-Angelli,Vishal Sanchania,Daisuke Kiga,Natalie G Farny,Christopher T Workman

doi:10.1038/s42003-020-01127-5

Jacob Beal, Markus Gershater + Show 10 more

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https://doi.org/10.1038/s42003-020-01127-5

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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.

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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
It is desirable to involve a large diversity of instruments and laboratories, such as those participating in the International Genetically Engineered Machines competition[8], where hundreds of teams at the high school, undergraduate, and graduate levels have been organized previously to study reproducibility and calibration for fluorescence measurements in engineered E. coli[9,10]
We organized a large-scale interlaboratory study within International Genetically Engineered Machines (iGEM) to compare three candidate OD calibration protocols: a colony-forming unit (CFU) assay, the de facto standard assay for determining viable cell count; comparison with colloidal silica (LUDOX) and water, previously used for normalizing fluorescence measurements[9]; and serial dilution of silica microspheres, a new protocol based on a recent study of microbial growth[7]

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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. We organized a large-scale interlaboratory study within iGEM to compare three candidate OD calibration protocols: a colony-forming unit (CFU) assay, the de facto standard assay for determining viable cell count; comparison with colloidal silica (LUDOX) and water, previously used for normalizing fluorescence measurements[9]; and serial dilution of silica microspheres, a new protocol based on a recent study of microbial growth[7]. Adoption of this recommendation is expected to enable effective use of OD data for estimation of cell count, comparison of plate reader measurements with single-cell measurements such as flow cytometry, improved replicability, and better cross-laboratory comparison of data

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