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Robust Estimation of Bacterial Cell Count from Optical Density

Beal, Jacob and Farny, Natalie G. and Haddock-Angelli, Traci and Selvarajah, Vinoo and Baldwin, Geoff S. and Buckley-Taylor, Russell and Gershater, Markus and Kiga, Daisuke and Marken, John and Sanchania, Vishal and Sison, Abigail and Workman, Christopher T. (2019) Robust Estimation of Bacterial Cell Count from Optical Density. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20191014-134916544

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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.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/803239DOIDiscussion Paper
ORCID:
AuthorORCID
Beal, Jacob0000-0002-1663-5102
Marken, John0000-0001-9696-088X
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. bioRxiv preprint first posted online Oct. 13, 2019. Partial support for this work was provided by NSF Expeditions in Computing Program Award #1522074 as part of the Living Computing Project. This document does not contain technology or technical data controlled under either the U.S. International Traffic in Arms Regulations or the U.S. Export Administration Regulations. Author Contributions: • Conceptualization: J.B., N.G.F., T.H-A., V.S.-1, G.S.B., R.B-T., M.G., D.K., J.M., C.T.W. • Data curation: J.B., N.G.F., T.H-A., V.S.-1 • Formal analysis: J.B. • Investigation: Experimental data gathered by iGEM Interlab Study Contributors (all authors listed in Supplementary Note: iGEM Interlab Study Contributors) • Methodology: J.B., N.G.F., T.H-A., V.S.-1, G.S.B., R.B-T., M.G., D.K., J.M., V.S.-2, A.S., C.T.W. • Project administration: J.B., N.G.F., T.H-A. • Resources: T.H-A., V.S.-1, A.S. • Software: J.B. • Writing (original draft): J.B., N.G.F. • Writing (review & editing): J.B., N.G.F., T.H-A., G.S.B., J.M., C.T.W., V.S.-2. The authors declare no competing interests.
Funders:
Funding AgencyGrant Number
NSFCCF-1522074
Record Number:CaltechAUTHORS:20191014-134916544
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20191014-134916544
Official Citation:Robust Estimation of Bacterial Cell Count from Optical Density. Jacob Beal, Natalie Gilks Farny, Traci Haddock-Angelli, Vinoo Selvarajah, Geoff S. Baldwin, Russell Buckley-Taylor, Markus Gershater, Daisuke Kiga, John P Marken, Vishal Sanchania, Abigail Sison, Christopher Workman, iGEM Interlab Study Contributors. bioRxiv 803239; doi: https://doi.org/10.1101/803239
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:99256
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:14 Oct 2019 21:06
Last Modified:14 Oct 2019 21:06

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