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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. In an inter-laboratory study, the authors compare the accuracy and performance of three optical density calibration protocols (colloidal silica, serial dilution of silica microspheres, and colony-forming unit (CFU) assay). They demonstrate that serial dilution of silica microspheres is the best of these tested protocols, allowing precise and robust calibration that is easily assessed for quality control and can also evaluate the effective linear range of an instrument. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]