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Robust Estimation of Bacterial Cell Count from Optical Density

View ORCID ProfileJacob Beal, Natalie G. Farny, Traci Haddock-Angelli, Vinoo Selvarajah, Geoff S. Baldwin, Russell Buckley-Taylor, Markus Gershater, Daisuke Kiga, John Marken, Vishal Sanchania, Abigail Sison, Christopher T. Workman, the iGEM Interlab Study Contributors
doi: https://doi.org/10.1101/803239
Jacob Beal
1Raytheon BBN Technologies, Cambridge, MA, USA
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  • For correspondence: jakebeal@ieee.org
Natalie G. Farny
2Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA, USA
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Traci Haddock-Angelli
3iGEM Foundation, Cambridge, MA, USA
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Vinoo Selvarajah
3iGEM Foundation, Cambridge, MA, USA
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Geoff S. Baldwin
4Department of Life Sciences and IC-Centre for Synthetic Biology, Imperial College London, London, UK
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Russell Buckley-Taylor
4Department of Life Sciences and IC-Centre for Synthetic Biology, Imperial College London, London, UK
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Markus Gershater
5Synthace, London, UK
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Daisuke Kiga
6Faculty of Science and Engineering, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan
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John Marken
7Department of Bioengineering, California Institute of Technology, Pasadena, CA, USA
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Vishal Sanchania
5Synthace, London, UK
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Abigail Sison
3iGEM Foundation, Cambridge, MA, USA
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Christopher T. Workman
8DTU-Bioengineering, Technical University of Denmark, Kongens Lyngby, Denmark
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Abstract

Optical density (OD) is a fast, cheap, and high-throughput measurement widely used to estimate the density of cells in liquid culture. These measurements, however, cannot be compared between instruments without a standardized calibration protocol and are challenging to relate to actual cell count. We address these shortcomings with an interlaboratory study comparing three OD calibration protocols, as applied to eight strains of E. coli engineered to constitutively express varying levels of GFP. These three protocols—comparison with colloidal silica (LUDOX), serial dilution of silica microspheres, and a reference colony-forming unit (CFU) assay—are all simple, low-cost, and highly accessible. Based on the results produced by the 244 teams completing this interlaboratory study, we recommend calibrating OD using serial dilution of silica microspheres, which readily produces highly precise calibration (95.5% of teams having residuals less than 1.2-fold), is easily assessed for quality control, and as a side effect also assesses the effective linear range of an instrument. Moreover, estimates of cell count from silica microspheres can be combined with fluorescence calibration against fluorescein to obtain units of Molecules of Equivalent Fluorescein (MEFL), allowing direct comparison and data fusion with equivalently calibrated 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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Posted October 13, 2019.
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Robust Estimation of Bacterial Cell Count from Optical Density
Jacob Beal, Natalie G. Farny, Traci Haddock-Angelli, Vinoo Selvarajah, Geoff S. Baldwin, Russell Buckley-Taylor, Markus Gershater, Daisuke Kiga, John Marken, Vishal Sanchania, Abigail Sison, Christopher T. Workman, the iGEM Interlab Study Contributors
bioRxiv 803239; doi: https://doi.org/10.1101/803239
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Robust Estimation of Bacterial Cell Count from Optical Density
Jacob Beal, Natalie G. Farny, Traci Haddock-Angelli, Vinoo Selvarajah, Geoff S. Baldwin, Russell Buckley-Taylor, Markus Gershater, Daisuke Kiga, John Marken, Vishal Sanchania, Abigail Sison, Christopher T. Workman, the iGEM Interlab Study Contributors
bioRxiv 803239; doi: https://doi.org/10.1101/803239

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