title: Probing phenotypic growth in expanding Bacillus subtilis biofilms.
abstract: We develop an optical imaging technique for spatially and temporally tracking biofilm growth and the distribution of the main phenotypes of a Bacillus subtilis strain with a triple-fluorescent reporter for motility, matrix production, and sporulation. We develop a calibration procedure for determining the biofilm thickness from the transmission images, which is based on Beer-Lambert's law and involves cross-sectioning of biofilms. To obtain the phenotype distribution, we assume a linear relationship between the number of cells and their fluorescence and determine the best combination of calibration coefficients that matches the total number of cells for all three phenotypes and with the total number of cells from the transmission images. Based on this analysis, we resolve the composition of the biofilm in terms of motile, matrix-producing, sporulating cells and low-fluorescent materials which includes matrix and cells that are dead or have low fluorescent gene expression. We take advantage of the circular growth to make kymograph plots of all three phenotypes and the dominant phenotype in terms of radial distance and time. To visualize the nonlocal character of biofilm growth, we also make kymographs using the local colonization time. Our technique is suitable for real-time, noninvasive, quantitative studies of the growth and phenotype distribution of biofilms which are either exposed to different conditions such as biocides, nutrient depletion, dehydration, or waste accumulation.
authors.affiliation: School of Mechanical Engineering, University of Science and Technology Beijing, Beijing, 100083, China. xiaoling@me.ustb.edu.cn.|School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA. xiaoling@me.ustb.edu.cn.
authors.forename: Xiaoling
authors.identifier: http://orcid.org/0000-0003-1659-466X
authors.initials: X
authors.lastname: Wang
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Koehler
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA.|Department of Chemical and Biological Engineering, Montana State University, Bozeman, MT, 59717-3920, USA.
authors.forename: James N
authors.initials: JN
authors.lastname: Wilking
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA.
authors.forename: Naveen N
authors.initials: NN
authors.lastname: Sinha
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 02138, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Cabeen
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA.
authors.forename: Siddarth
authors.initials: S
authors.lastname: Srinivasan
authors.affiliation: CNRS, LPMC UMR 7336, Université Nice Sophia Antipolis, Parc Valrose, 06108, Nice, France.
authors.forename: Agnese
authors.initials: A
authors.lastname: Seminara
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA.
authors.forename: Shmuel
authors.initials: S
authors.lastname: Rubinstein
authors.affiliation: Department of Mechanical Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, China.
authors.forename: Qingping
authors.initials: Q
authors.lastname: Sun
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Brenner
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA. weitz@seas.harvard.edu.|Department of Physics, Harvard University, Cambridge, MA, 02138, USA. weitz@seas.harvard.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Weitz
mesh_terms: D001412:Bacillus subtilis / Q000254:growth & development*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development*; D003470:Culture Media / Q000737:chemistry; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008962:Models, Theoretical; D061848:Optical Imaging / Q000379:methods*; D010641:Phenotype
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media
keywords: Bacillus subtilis; Biofilm; Growth characterization; Phenotype switching; Phenotype visualization