Applied Microbiology and Biotechnology

, Volume 100, Issue 10, pp 4607–4615 | Cite as

Probing phenotypic growth in expanding Bacillus subtilis biofilms

  • Xiaoling WangEmail author
  • Stephan A. Koehler
  • James N. Wilking
  • Naveen N. Sinha
  • Matthew T. Cabeen
  • Siddarth Srinivasan
  • Agnese Seminara
  • Shmuel Rubinstein
  • Qingping Sun
  • Michael P. Brenner
  • David A. WeitzEmail author
Methods and protocols


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.


Bacillus subtilis Phenotype visualization Growth characterization Biofilm Phenotype switching 



We thank Richard Losick’s group and Roberto Kolter’s group for providing triple-fluorescent-labeled Bacillus subtilis strains. This work was supported by the National Science Foundation (DMR-1310266, DMS-1411694), the Harvard Materials Research Science and Engineering Center (DMR-1420570), the National Natural Science Foundation of China (11272002), and the Beijing Higher Education Young Elite Teacher Project (YETP0363).

Compliance with ethical standards


The National Science Foundation (DMR-1310266, DMS-1411694), the Harvard Materials Research Science and Engineering Center (DMR-1420570), the National Natural Science Foundation of China (11272002), and the Beijing Higher Education Young Elite Teacher Project (YETP0363).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7461_MOESM1_ESM.pdf (83 kb)
ESM 1 (PDF 83 kb)
253_2016_7461_MOESM2_ESM.avi (1.9 mb)
ESM 2 (AVI 1930 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Xiaoling Wang
    • 1
    • 2
    Email author
  • Stephan A. Koehler
    • 2
  • James N. Wilking
    • 2
    • 3
  • Naveen N. Sinha
    • 2
  • Matthew T. Cabeen
    • 4
  • Siddarth Srinivasan
    • 2
  • Agnese Seminara
    • 5
  • Shmuel Rubinstein
    • 2
  • Qingping Sun
    • 6
  • Michael P. Brenner
    • 2
  • David A. Weitz
    • 2
    • 7
    Email author
  1. 1.School of Mechanical EngineeringUniversity of Science and Technology BeijingBeijingChina
  2. 2.School of Engineering and Applied SciencesHarvard UniversityCambridgeUSA
  3. 3.Department of Chemical and Biological EngineeringMontana State UniversityBozemanUSA
  4. 4.Department of Molecular and Cellular BiologyHarvard UniversityCambridgeUSA
  5. 5.CNRS, LPMC UMR 7336, Université Nice Sophia AntipolisNiceFrance
  6. 6.Department of Mechanical EngineeringThe Hong Kong University of Science and TechnologyHong Kong SARChina
  7. 7.Department of PhysicsHarvard UniversityCambridgeUSA

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