Robust estimation of bacterial cell count from optical density
Jacob Beal, Natalie G. Farny, Traci Haddock-Angelli, Vinoo Selvarajah, Geoff Baldwin, Russell Buckley-Taylor, Markus Gershater, Daisuke Kiga, John P. Marken, Vishal Sanchania, Abigail Sison, Christopher T. Workman, iGEM Interlab Study Contributors, Aachen, Meryem Pehlivan, Biel Badia Roige, Aalto-Helsinki, Tiu Aarnio, Samu Kivistö, Jessica Koski, L. Lehtonen, Denise Pezzutto, Pauliina Rautanen, AHUT_China, Weixin Bian, Zhiyuan Hu, Zhihao Liu, Zi Liu, Liang Ma, Luyao Pan, Zichen Qin, Huichao Wang, Xiangxuan Wang, Hao Xu, Xia Xu, Aix-Marseille, Yorgo El Moubayed, ASTWS-China, Shan Dong, Choco Fang, Hanker He, Henry He, Fang‐Liang Huang, Ruyi Shi, Cassie Tang, Christian Tang, Shirly Xu, Calvin Yan, Athens, Natalia Bartzoka, Eleni Kanata, María Kapsokefalou, Xanthi-Lida Katopodi, Eleni Kostadima, Ioannis V. Kostopoulos, Stylianos Kotzastratis, Antonios Ε. Koutelidakis, Vasilios Krokos, Maria Litsa, Ioannis Ntekas, Panagiotis M. Spatharas, Ourania Tsitsilonis, Anastasia Zerva, Austin_LASA, Vidhya Annem, Eli Cone, Noel Elias, Shreya Gupta, Kendrick Lam, Anna Tutuianu, Austin_UTexas, Dennis M. Mishler, Bibiana Toro, Baltimore_BioCrew, Akinwumi Akinfenwa, Frank Burns, Heydy Herbert, M. Gail Jones, Sarah Laun, Shikei Morrison, Zion Smith, BCU, Peng Zhao, Zhou Ziwei, BFSUICC-China, Rui Deng, Yilin Huang, Tingyue Li, Yingqi Ma, Zhiyuan Shen, Chenxi Wang, Yuyao Wang, Tianyan Zhao, BGIC-Global, Yusen Lang, Yuteng Liang, Xueyao Wang, Yi Wu, BGU_Israel, Dror Aizik
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.