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Cited by 290bioluminescence and chemiluminescence researchCell Image Analysis TechniquesScientific Measurement and Uncertainty Evaluation

Robust estimation of bacterial cell count from optical density

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Beal, Jacob, Farny, Natalie G., Haddock-Angelli, Traci, Selvarajah, Vinoo, Baldwin, Geoff S., Buckley-Taylor, Russell, Gershater, Markus, Kiga, Daisuke +1645 more·2020·Communications Biology
DOI 10.1038/s42003-020-01127-5

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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.

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Beal, J., Farny, N. G., Haddock-Angelli, T., Selvarajah, V., Baldwin, G. S., Buckley-Taylor, R., Gershater, M., Kiga, D., Marken, J., Sanchania, V., Sison, A., Workman, C. T., Contributors, I. I. S., Aachen, Pehlivan, M., Roige, B. B., Aalto-Helsinki, Aarnio, T., Kivisto, S., Koski, J., Lehtonen, L., Pezzutto, D., Rautanen, P., AHUT_China, Bian, W., Hu, Z., Liu, Z., Liu, Z., Ma, L., Pan, L., Qin, Z., Wang, H., Wang, X., Xu, H., Xu, X., Aix-Marseille, El Moubayed, Y., ASTWS-China, Dong, S., Fang, C., He, H., He, H., Huang, F., Shi, R., Tang, C., Tang, C., Xu, S., Yan, C., Athens, Bartzoka, N., Kanata, E., Kapsokefalou, M., Katopodi, X.-L., Kostadima, E., Kostopoulos, I. V., Kotzastratis, S., Koutelidakis, A. E., Krokos, V., Litsa, M., Ntekas, I., Spatharas, P., Tsitsilonis, O. E., Zerva, A., Austin_LASA, Annem, V., Cone, E., Elias, N., Gupta, S., Lam, K., Tutuianu, A., Austin_UTexas, Mishler, D. 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S., Thormar, E. A., Uslu, A., leelahakorn, N., UESTC-China, Ding, S., Li, C., Tan, H., Xu, Y., Yang, J., UFlorida, Gamoneda, D., Kantor, N., Trujillo-Rodriguez, L., Turner, M., UGA, George, S., McConnell, K., Pollitt, C., UI_Indonesia, Amin, I. F., Ikhsan, M., Japranata, V. V., Laurentius, A., Nurachman, L. A., Pratama, M. I. A., UiOslo_Norway, Dirven, Y., Frohlich, L., Linke, D., Mertes, V., Rolfsnes, R. R., Saragliadis, A., UIOWA, Castillo, S., Chinnathambi, S., Ellermeier, C., Farrell, J., Fassler, J., Fuentes, E., Ryan, S., Sander, E., UIUC_Illinois, Bott, A., Healy, L., Karumanchi, P., Ruzicka, A., Wang, Z., ULaval, Byatt, G., Despres, P. C., Dube, A., Lemieux, P., Echelard, F., Lortie, L.-A., Rouleau, F. D., ULaVerne_Collab, Barrington, S., Basulto, C., Delgadillo, S., Leon, K. D., Madrid, M., Meas, C., Sabandal, A., Sanchez, M. A., Tsui, J., Woodward, N., UMaryland, Battina, R., Boyer, J., Cherupalla, A., Chiang, J., Heng, M., Keating, C., Liang, T., Loke, C. K., Premo, J., Srinivasan, K., Starkel, J., Zheng, D., UNebraska-Lincoln, Astorino, G., Cott, R. V., Guo, J., Kortus, D., Niu, W., Unesp_Brazil, Freire, P. J. C., Pedrolli, D. B., Ribeiro, N. V., Silva, B. F., Vanini, N. V., Mota, M. S. D., Crispim, L. D. S., UNSW_Australia, Chapman, T., Gaitt, T., Jones, M., Watson, E., UPF_CRG_Barcelona, Lopez-Grado, G., Sans, L., UPF_CRG_Barcelona, Brink, M., Rajagopal, V., Ramstrom, E., US_AFRL_CarrollHS, Bete, A., Camacho, Y., Carter, J., Davis, C., Dong, J., Ehrenworth, A., Goodson, M., Guptil, C., Herrmann, M., Hung, C., Jesse, H., Krabacher, R., McDonald, D., Menart, P., O’Leary, T., Polanka, L., Poole, A., Varaljay, V., USMA-West_Point, Appel, A., Cave, J., Huuki, L., McDonough, M., Mills, C., Mitropoulos, A., Pruneski, J., Wickiser, K., USP-Brazil, Buson, F. X., Flores, V., Lima, G. M., Rosa, C. G. T., UST_Beijing, Bao, G., Dong, H., Luo, Z., Peng, J., USTC, An, Y., Cheng, C., Jiang, Z., Kong, L., Luo, C., Luo, L., Shi, Y., Tang, E., Wang, P., Wang, Y., Xu, G., Yu, W., Zhang, B., Zhang, Q., UT-Knoxville, Garcia, D., Jiang, N., Kristy, B., Laurel, R., Leitner, K., Loeffler, F., Ripp, S., Street, M., Utrecht, Amheine, K., Bindt, F., Boer, M., Boxem, M., Govers, J., Jongkees, S., Pattiradjawane, L., Swart, P., Tsang, H., Graaf, F. D., ten Dam, M., Heijningen, F. V., Valencia_UPV, Boada, Y., Vignoni, A., Vilnius-Lithuania, Brasas, V., Gaizauskaite, A., Jakutis, G., Jasiunas, S., Juskaite, I., Ritmejeris, J., Vaitkus, D., Venclovas, T., Vitkute, K., Yeliseyeva, H., Zukauskaite, K., Zvirblyte, J., Vilnius-Lithuania-OG, Karpus, L., Mazelis, I., Rokaitis, I., Virginia, Akingbesote, N. D., Culfogienis, D., Huang, W., Park, K., Warwick, Ahuja, J., Corre, C., Dhaliwal, G., Evans, R., Hill, K., Holman, O., Jaramillo, A., Khalid, A., Lawrence, J., Mansfield, L., O’Brien, J., Ong, J., Prakash, S., Whiteside, J., Washington, Anderson, K., Chun, E., Kim, G., Nguyen, A., Shola, C., Toghani, D., Wong, A., Wong, J., Yung, J., WashU_StLouis, Johnson, E., Lahad, D., Nicholson, K., Pedamallu, H., Phelan, C., Waterloo, Fikry, C., Fulton, L., Lassel, N., Perera, D., Robin, M., Shaw, N., Westminster_UK, Bowman, K., Coleman, S., Emilov, K., Gaspar, C., Jenakendran, J., Mubeen, S., Obrvan, M., Smith, C., WHU-China, Bo, T., Liaoqi, D., Tianyi, C., Yuan, X., Yue, Q., William_and_Mary, Do, S., Fang, X., Jones, E., Laury, J., Liu, W., Oliver, A., Parr, L., Saha, M., Shen, C., Son, T., Urban, J., Verma, Y., Zhou, H., Worldshaper-Wuhan, Dong, S., Hao, Z., Kuang, Y., Liu, T., Zhou, R., WPI_Worcester, Arruda, B., Farny, N., Hao, M., Pearce, C., Rebello, A., Sharma, A., Sumner, K., Sweet, B., XJTLU-CHINA, Lin, J., XJTU-China, Mengtao, D., Peiyao, F., Xinlei, F., XMU-China, Cai, N., Chen, J., Fu, Y., Hu, Y., Qiang, Y., Wang, Q., Yang, R., Yucheng, C., Zheng, J., Yale, Chang, K., Gao, C., Isaacs, F., Li, K., Moscoso, R., Patel, J., Telesz, L., Tirad, A., ZJU-China, Cao, Q., Feng, X., Lu, Y., Zhang, X., Zhou, X., ZJUT-China, Sun, D., Yuan, Z., & Zhou, J.
Affiliations
  • Imperial College London (GB)
  • Synthace (United Kingdom) (GB)
  • Waseda University (JP)
  • California Institute of Technology (US)
  • Technical University of Denmark (DK)
  • RWTH Aachen University (DE)
  • École Polytechnique Fédérale de Lausanne (CH)
  • University of Helsinki (FI)
  • Anhui University of Technology (CN)
  • Nanjing University (CN)
  • Shanghai Jiao Tong University (CN)
  • South China University of Technology (CN)
  • Stony Brook University (US)
  • South China Agricultural University (CN)
  • BGI Group (China) (CN)
  • China Pharmaceutical University (CN)
  • Stony Brook School (US)
  • Nanjing Agricultural University (CN)
  • National Technical University of Athens (GR)
  • University of British Columbia (CA)
  • Beijing City University (CN)
  • Beijing Foreign Studies University (CN)
  • Bielefeld University (DE)
  • Institut Químic de Sarrià (ES)
  • Ben-Gurion University of the Negev (IL)
  • CHA University (KR)
  • Beijing Institute of Technology (CN)
  • Georgia State University (US)
  • Beijing National Day School (CN)
  • Beijing Normal University (CN)
  • Boston University (US)
  • National Chung Cheng University (TW)
  • University of Calgary (CA)
  • Fudan University (CN)
  • Cardiff University (GB)
  • Chalmers University of Technology (SE)
  • Carnegie Mellon University Qatar (QA)
  • Jiangnan University (CN)
  • Columbia University (US)
  • University of Electronic Science and Technology of China (CN)
  • Cornell University (US)
  • Central South University (CN)
  • Dalian University of Technology (CN)
  • National Tsing Hua University (TW)
  • Colorado School of Mines (US)
  • San Diego Unified School District (US)
  • Universidad de las Fuerzas Armadas ESPE (EC)
  • East China University of Science and Technology (CN)
  • Emory University (US)
  • ETH Zurich (CH)
  • University of Exeter (GB)
  • Friedrich-Alexander-Universität Erlangen-Nürnberg (DE)
  • Fujian Normal University (CN)
  • Gifu University (JP)
  • Université Paris-Saclay (FR)
  • University of Groningen (NL)
  • Hankuk University of Foreign Studies (KR)
  • Universität Hamburg (DE)
  • Hubei University of Technology (CN)
  • Hebrew University of Jerusalem (IL)
  • Indian Institute of Science Bangalore (IN)
  • University of Hong Kong (HK)
  • Hong Kong University of Science and Technology (HK)
  • Indian Institute of Science Education and Research Kolkata (IN)
  • Institute of Chemical Technology (IN)
  • Indian Institute of Science Education and Research, Bhopal (IN)
  • Indian Institute of Science Education and Research Mohali (IN)
  • Indian Institute of Technology Delhi (IN)
  • Indian Institute of Technology Kanpur (IN)
  • Indian Institute of Technology Madras (IN)
  • Jilin University (CN)
  • Chinese University of Hong Kong (HK)
  • University of Würzburg (DE)
  • Kanagawa Institute of Technology (JP)
  • University of Science and Technology of China (CN)
  • Sun Yat-sen University (CN)
  • King's College London (GB)
  • Korea University (KR)
  • Leiden University (NL)
  • University of Lethbridge (CA)
  • Texas Tech University (US)
  • Lund University (SE)
  • Universidad Complutense de Madrid (ES)
  • Philipps University of Marburg (DE)
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  • Middle East Technical University (TR)
  • University of Michigan (US)
  • University of Minnesota (US)
  • Université de Montpellier (FR)
  • Technical University of Munich (DE)
  • University of Graz (AT)
  • National Chung Hsing University (TW)
  • National Yang Ming Chiao Tung University (TW)
  • Northeastern University (CN)
  • Newcastle University (GB)
  • Northwestern University (US)
  • University of Nottingham (GB)
  • Northwestern Polytechnical University (CN)
  • Norwegian University of Science and Technology (NO)
  • Nanyang Technological University (SG)
  • Nazarbayev University (KZ)
  • National University of Defense Technology (CN)
  • National University of Singapore (SG)
  • Northwest University (CN)
  • National Yang Ming University (TW)
  • New York University Abu Dhabi (AE)
  • University of Oxford (GB)
  • Learning Planet Institute (FR)
  • Institut Pasteur (FR)
  • Second Military Medical University (CN)
  • Peking University (CN)
  • University of Pittsburgh (US)
  • Purdue University West Lafayette (US)
  • Nankai University (CN)
  • Queen's University (CA)
  • Rajalakshmi Engineering College (IN)
  • Rose–Hulman Institute of Technology (US)
  • Sichuan University (CN)
  • Ocean University of China (CN)
  • ShanghaiTech University (CN)
  • Shandong University (CN)
  • Sorbonne Université (FR)
  • University of St Andrews (GB)
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  • Rhode Island School of Design (US)
  • University of Stuttgart (DE)
  • University of Chicago (US)
  • Shenzhen University (CN)
  • Tecnológico de Monterrey (MX)
  • Aristotle University of Thessaloniki (GR)
  • Tongji University (CN)
  • University of Toronto (CA)
  • Université Toulouse III - Paul Sabatier (FR)
  • Technische Universität Darmstadt (DE)
  • Eindhoven University of Technology (NL)
  • Tianjin University of Science and Technology (CN)
  • University of Alberta (CA)
  • University of California, Davis (US)
  • University of California San Diego (US)
  • University of Chinese Academy of Sciences (CN)
  • University of Chile (CL)
  • University College of Arts Crafts and Design (SE)
  • University College London (GB)
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  • University of Florida (US)
  • University of Georgia (US)
  • University of Indonesia (ID)
  • University of Oslo (NO)
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  • Université Laval (CA)
  • University of La Verne (US)
  • University of Maryland, College Park (US)
  • University of Nebraska–Lincoln (US)
  • UNSW Sydney (AU)
  • Universitat Pompeu Fabra (ES)
  • Uppsala University (SE)
  • Dayton School District (US)
  • United States Military Academy (US)
  • Universidade de São Paulo (BR)
  • University of Science and Technology Beijing (CN)
  • University of Tennessee at Knoxville (US)
  • Utrecht University (NL)
  • Universitat Politècnica de València (ES)
  • Vilnius University (LT)
  • University of Virginia (US)
  • University of Warwick (GB)
  • University of Waterloo (CA)
  • University of Westminster (GB)
  • Wuhan University (CN)
  • William & Mary (US)
  • Worcester Polytechnic Institute (US)
  • Xiamen University (CN)
  • Yale University (US)
  • Zhejiang University of Technology (CN)
  • Universidade Estadual Paulista (Unesp) (BR)
  • University of Washington (US)
  • Washington University in St. Louis (US)
  • Xi’an Jiaotong-Liverpool University (CN)
  • Xi'an Jiaotong University (CN)
  • Zhejiang University (CN)
Corresponding author
  • Beal, Jacob
  • Baldwin, Geoff S.
  • Gershater, Markus
  • Workman, Christopher T.
ORCID
Venue
Communications Biology
Publisher
Springer Science and Business Media LLC
Published
2020-09-17
Volume
3
Issue
1
Pages
512-512
ISSN
2399-3642 (ISSN-L)
DOI
10.1038/s42003-020-01127-5
OpenAlex
W3087475959
Cited by
290
References
1
Citations by year
2026: 54 · 2025: 65 · 2024: 66 · 2023: 43 · 2022: 26 · 2021: 31 · 2020: 3 · 2012: 1
FWCI
8.35
Type
journal article
Language
en
Update notices
correction (2020-10-27)
Open access
Licence
Locations
  • Communications Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • Nazarbayev University Repository (Nazarbayev University) (submittedVersion, other-oa)
  • Leiden Repository (Leiden University) (submittedVersion, cc-by)
  • Chalmers Research (Chalmers University of Technology) (submittedVersion)
  • CaltechAUTHORS (California Institute of Technology) (submittedVersion, cc-by)
  • UCL Discovery (University College London) (submittedVersion)
  • Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion, cc-by)
  • St Andrews Research Repository (St Andrews Research Repository) (submittedVersion, cc-by)
  • NORA - Norwegian Open Research Archives (submittedVersion, cc-by)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion, cc-by)
Funders
National Science Foundation (1522074)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
  • Social Sciences › Decision Sciences › Statistics, Probability and Uncertainty › Scientific Measurement and Uncertainty Evaluation
Keywords
silica microspheres, serial dilution, interlaboratory comparison, plate reader, quality control
MeSH terms
Calibration, Cell Count, Escherichia coli, Flow Cytometry, Fluorescence, Gene Expression Regulation, Bacterial, Bacterial Load

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