Single-Cell Analysis of Mycobacteria Using Microfluidics and Time-Lapse Microscopy

  • Neeraj DharEmail author
  • Giulia Manina
Part of the Methods in Molecular Biology book series (MIMB, volume 1285)


The crucial role of phenotypic heterogeneity in bacterial physiology and adaptive responses has required the introduction of new ways to investigate bacterial individuality. Time-lapse microscopy is a powerful technique for evaluating phenotypic diversity in bacteria at the single-cell level, whether exploring the dynamics of gene expression and protein localization or characterizing the heterogeneous phenotypic response to perturbations. Here, we present protocols to carry out time-lapse imaging of mycobacteria at the single-cell level using either agarose pads or customized microfluidic devices. The sequences of images obtained can be analyzed using programs such as ImageJ and allow the investigator not only to extract various parameters of growth and gene expression dynamics but also to unravel the physiological basis behind phenomenon such as persistence against stresses.

Key words

Single-cell Time-lapse Microscopy Microfluidics Mycobacterium Cell growth Cell division Fluorescence Gene expression Single-cell analysis 


  1. 1.
    Avery SV (2006) Microbial cell individuality and the underlying sources of heterogeneity. Nat Rev Microbiol 4:577–587CrossRefPubMedGoogle Scholar
  2. 2.
    Dhar N, McKinney JD (2007) Microbial phenotypic heterogeneity and antibiotic tolerance. Curr Opin Microbiol 10:30–38CrossRefPubMedGoogle Scholar
  3. 3.
    Balaban NQ, Merrin J, Chait R et al (2004) Bacterial persistence as a phenotypic switch. Science 305:1622–1625CrossRefPubMedGoogle Scholar
  4. 4.
    Eldar A, Chary VK, Xenopoulos P et al (2009) Partial penetrance facilitates developmental evolution in bacteria. Nature 460:510–514PubMedPubMedCentralGoogle Scholar
  5. 5.
    Eldar A, Elowitz MB (2010) Functional roles for noise in genetic circuits. Nature 467:167–173CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Locke JC, Young JW, Fontes M et al (2011) Stochastic pulse regulation in bacterial stress response. Science 334:366–369CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Norman TM, Lord ND, Paulsson J et al (2013) Memory and modularity in cell-fate decision making. Nature 503:481–486CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Rotem E, Loinger A, Ronin I et al (2010) Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence. Proc Natl Acad Sci U S A 107:12541–12546CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Wakamoto Y, Dhar N, Chait R et al (2013) Dynamic persistence of antibiotic-stressed mycobacteria. Science 339:91–95CrossRefPubMedGoogle Scholar
  10. 10.
    Brehm-Stecher BF, Johnson EA (2004) Single-cell microbiology: tools, technologies, and applications. Microbiol Mol Biol Rev 68:538–559CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ackermann M, Stearns SC, Jenal U (2003) Senescence in a bacterium with asymmetric division. Science 300:1920CrossRefPubMedGoogle Scholar
  12. 12.
    de Jong IG, Beilharz K, Kuipers OP, Veening J-W (2011) Live cell imaging of Bacillus subtilis and Streptococcus pneumoniae using automated time-lapse microscopy. J Vis Exp 53:e3145Google Scholar
  13. 13.
    Golding I, Paulsson J, Zawilski SM et al (2005) Real-time kinetics of gene activity in individual bacteria. Cell 123:1025–1036CrossRefPubMedGoogle Scholar
  14. 14.
    Lindner AB, Madden R, Demarez A et al (2008) Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation. Proc Natl Acad Sci U S A 105:3076–3081CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Llopis PM, Jackson AF, Sliusarenko O et al (2010) Spatial organization of the flow of genetic information in bacteria. Nature 466:77–81CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Locke JCW, Elowitz MB (2009) Using movies to analyse gene circuit dynamics in single cells. Nat Rev Micro 7:383–392CrossRefGoogle Scholar
  17. 17.
    Stewart EJ, Madden R, Paul G et al (2005) Aging and death in an organism that reproduces by morphologically symmetric division. PLoS Biol 3:e45CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Wang P, Robert L, Pelletier J et al (2010) Robust growth of Escherichia coli. Curr Biol 20:1099–1103CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Young JW, Locke JC, Altinok A et al (2012) Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy. Nat Protoc 7:80–88CrossRefGoogle Scholar
  20. 20.
    Aldridge BB, Fernandez-Suarez M, Heller D et al (2012) Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic susceptibility. Science 335:100–104CrossRefPubMedGoogle Scholar
  21. 21.
    Golchin SA, Stratford J, Curry RJ et al (2012) A microfluidic system for long-term time-lapse microscopy studies of mycobacteria. Tuberculosis (Edinb) 92:489–496CrossRefGoogle Scholar
  22. 22.
    Joyce G, Williams KJ, Robb M et al (2012) Cell division site placement and asymmetric growth in mycobacteria. PLoS One 7:e44582CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Joyce G, Robertson BD, Williams KJ (2011) A modified agar pad method for mycobacterial live-cell imaging. BMC Res Notes 4:73Google Scholar
  24. 24.
    Makarov V, Manina G, Mikusova K et al (2009) Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science 324:801–804CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Sala C, Dhar N, Hartkoorn RC et al (2010) Simple model for testing drugs against nonreplicating Mycobacterium tuberculosis. Antimicrob Agents Chemother 54:4150–4158CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Santi I, Dhar N, Bousbaine D et al (2013) Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria. Nat Commun 4:2470. doi: 10.1038/ncomms3470 PubMedGoogle Scholar
  27. 27.
    Singh B, Nitharwal RG, Ramesh M et al (2013) Asymmetric growth and division in Mycobacterium spp.: compensatory mechanisms for non-medial septa. Mol Microbiol 88:64–76CrossRefPubMedGoogle Scholar
  28. 28.
    Vijay S, Nagaraja M, Sebastian J et al (2014) Asymmetric cell division in Mycobacterium tuberculosis and its unique features. Arch Microbiol 196:157–168CrossRefPubMedGoogle Scholar
  29. 29.
    Hett EC, Rubin EJ (2008) Bacterial growth and cell division: a mycobacterial perspective. Microbiol Mol Biol Rev 72:126–156CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Friend J, Yeo L (2010) Fabrication of microfluidic devices using polydimethylsiloxane. Biomicrofluidics 4:026502CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Weibel DB, Diluzio WR, Whitesides GM (2007) Microfabrication meets microbiology. Nat Rev Micro 5:209–218CrossRefGoogle Scholar
  32. 32.
    Whitesides G, Ostuni E, Takayama S et al (2001) Soft lithography in biology and biochemistry. Annu Rev Biomed Eng 3:335–373CrossRefPubMedGoogle Scholar
  33. 33.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682CrossRefPubMedGoogle Scholar
  34. 34.
    de Chaumont F, Dallongeville S, Chenouard N et al (2012) Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods 9:690–696CrossRefPubMedGoogle Scholar
  35. 35.
    Carpenter AE, Jones TR, Lamprecht MR et al (2006) Cell profiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7:R100CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Sliusarenko O, Heinritz J, Emonet T et al (2011) High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics. Mol Microbiol 80:612–627CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Wang Q, Niemi J, Tan C-M et al (2010) Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy. Cytometry A 77:101–110PubMedPubMedCentralGoogle Scholar
  38. 38.
    Patino S, Alamo L, Cimino M et al (2008) Autofluorescence of mycobacteria as a tool for detection of Mycobacterium tuberculosis. J Clin Microbiol 46:3296CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Laboratory of Microbiology and Microsystems, School of Life SciencesSwiss Federal Institute of Technology in Lausanne (EPFL)LausanneSwitzerland

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