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 (US) |
|---|---|
| Article number | 512 |
| Journal | Communications biology |
| Volume | 3 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 2020 |
| Externally published | Yes |
Bibliographical note
Publisher Copyright:© 2020, The Author(s).
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In: Communications biology, Vol. 3, No. 1, 512, 12.2020.
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TY - JOUR
T1 - Robust estimation of bacterial cell count from optical density
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AU - Beal, Jacob
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N1 - Publisher Copyright: © 2020, The Author(s).
PY - 2020/12
Y1 - 2020/12
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 - http://www.scopus.com/inward/record.url?scp=85091192744&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85091192744&partnerID=8YFLogxK
U2 - 10.1038/s42003-020-01127-5
DO - 10.1038/s42003-020-01127-5
M3 - Article
C2 - 32943734
AN - SCOPUS:85091192744
SN - 2399-3642
VL - 3
JO - Communications biology
JF - Communications biology
IS - 1
M1 - 512
ER -