Robust estimation of bacterial cell count from optical density

iGEM Interlab Study Contributors; Clarissa Melo Czekster; Simon John Powis

doi:10.1038/s42003-020-01127-5

Robust estimation of bacterial cell count from optical density

iGEM Interlab Study Contributors, Clarissa Melo Czekster, Simon John Powis

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Article number	512
Number of pages	29
Journal	Communications Biology
Volume	3
DOIs	https://doi.org/10.1038/s42003-020-01127-5
Publication status	Published - 17 Sept 2020

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@article{d7597d0f7eed452c9fe84fa2c7cc52fa,

title = "Robust estimation of bacterial cell count from optical density",

abstract = "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.",

author = "{iGEM Interlab Study Contributors} and Jacob Beal and Farny, {Natalie G.} and Traci Haddock-Angelli and Vinoo Selvarajah and Baldwin, {Geoff S.} and Russell Buckley-Taylor and Markus Gershater and Daisuke Kiga and John Marken and Vishal Sanchania and Abigail Sison and Workman, {Christopher T.} and {Melo Czekster}, Clarissa and Powis, {Simon John}",

note = "Partial support for this work was provided by NSF Expeditions in Computing Program Award #1522074 as part of the Living Computing Project, and by the Engineering and Physical Sciences Research Council [EP/R034915/1] and EU H2020 [820699]. ",

year = "2020",

month = sep,

day = "17",

doi = "10.1038/s42003-020-01127-5",

language = "English",

volume = "3",

journal = "Communications Biology",

issn = "2399-3642",

publisher = "Springer Nature",

}

TY - JOUR

T1 - Robust estimation of bacterial cell count from optical density

AU - iGEM Interlab Study Contributors

AU - Beal, Jacob

AU - Farny, Natalie G.

AU - Haddock-Angelli, Traci

AU - Selvarajah, Vinoo

AU - Baldwin, Geoff S.

AU - Buckley-Taylor, Russell

AU - Gershater, Markus

AU - Kiga, Daisuke

AU - Marken, John

AU - Sanchania, Vishal

AU - Sison, Abigail

AU - Workman, Christopher T.

AU - Melo Czekster, Clarissa

AU - Powis, Simon John

N1 - Partial support for this work was provided by NSF Expeditions in Computing Program Award #1522074 as part of the Living Computing Project, and by the Engineering and Physical Sciences Research Council [EP/R034915/1] and EU H2020 [820699].

PY - 2020/9/17

Y1 - 2020/9/17

N2 - 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.

AB - 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.

UR - https://doi.org/10.1038/s42003-020-01371-9

U2 - 10.1038/s42003-020-01127-5

DO - 10.1038/s42003-020-01127-5

M3 - Article

SN - 2399-3642

VL - 3

JO - Communications Biology

JF - Communications Biology

M1 - 512

ER -

Robust estimation of bacterial cell count from optical density

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