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

• Published in: Nature protocols Vol. 6; no. 5; pp. 625 - 639
• Main Authors: Kong, Lingbo, Zhang, Pengfei, Wang, Guiwen, Yu, Jing, Setlow, Peter, Li, Yong-qing
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.05.2011; Nature Publishing Group
• Subjects: 631/1647/2204/2112; 631/1647/328; 631/1647/527/1821; 631/326/41/1969; Acids; Analytical Chemistry; Bacillus - physiology; Bacteria; Biological Techniques; Biomedical and Life Sciences; Chemical properties; Computational Biology/Bioinformatics; Data processing; Fluorescence; Fluorescence microscopy; Genetic aspects; Lasers; Life Sciences; Methods; Microarrays; Microscopes; Microscopy; Microscopy - methods; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Nucleic acids; Optical Tweezers; Organic Chemistry; Protocol; Raman spectroscopy; Spectroscopy; Spectrum analysis; Spectrum Analysis, Raman - methods; Spore germination; Spores (Bacteria); Spores, Bacterial - growth & development; Stains; United States; United States > US
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/nprot.2011.307

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.