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Characterization of bacterial spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers

Nature Protocols volume 6, pages 625–639 (2011)Cite this article

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Abstract

This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. The characterization consists of the following steps: (i) loading heat-activated dormant spores into a temperature-controlled microscope sample holder containing a germinant solution plus a nucleic acid stain; (ii) capturing a single spore with optical tweezers; (iii) simultaneously measuring phase-contrast images, Raman spectra and fluorescence images of the optically captured spore at 2- to 10-s intervals; and (iv) analyzing the acquired data for the loss of spore refractility, changes in spore-specific molecules (in particular, dipicolinic acid) and uptake of the nucleic acid stain. This information leads to precise correlations between various germination events, and takes 1–2 h to complete. The method can also be adapted to use multi-trap Raman spectroscopy or phase-contrast microscopy of spores adhered on a cover slip to simultaneously obtain germination parameters for multiple individual spores.

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Figure 1: Scheme of the experimental setup.
Figure 2: Phase-contrast image and Raman spectrum of an individual trapped dormant B. cereus spore.
Figure 3: Phase-contrast and Raman spectral images of multiple trapped dormant B. cereus spores.
Figure 4: Sample container and heating stage designed for temperature control.
Figure 5: Simultaneous recording of Raman spectra, phase-contrast images and fluorescence images of a single optically trapped B. cereus spore germinating at 24 °C with 1 mM L-alanine plus 0.
Figure 6: Germination heterogeneity characterized by time of Trelease.

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Acknowledgements

This work was supported by a grant from the Army Research Office (Y.-q.L. and P.S.) and by a Multidisciplinary University Research Initiative award from the United States Department of Defense (P.S. and Y.-q.L.).

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Authors and Affiliations

  1. Department of Physics, East Carolina University, Greenville, North Carolina, USA

    Lingbo Kong, Pengfei Zhang, Guiwen Wang, Jing Yu & Yong-qing Li

  2. Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut, USA

    Peter Setlow

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Contributions

L.K. and Y.-q.L. designed the experimental setup, performed the research, analyzed data and wrote the manuscript. P.Z., G.W. and J.Y. performed the experiments and analyzed data. P.S. provided spore samples, supervised the microbiological work and revised the manuscript.

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Correspondence to Yong-qing Li.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Code for the computer programs used in this protocol. QSI CCD control_VB code. DAQ control_Matlab code. (RTF 320 kb)

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Kong, L., Zhang, P., Wang, G. et al. Characterization of bacterial spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers. Nat Protoc 6, 625–639 (2011). https://doi.org/10.1038/nprot.2011.307

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