title: Characterization of bacterial spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers.
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.
authors.affiliation: Department of Physics, East Carolina University, Greenville, North Carolina, USA.
authors.forename: Lingbo
authors.initials: L
authors.lastname: Kong
authors.forename: Pengfei
authors.initials: P
authors.lastname: Zhang
authors.forename: Guiwen
authors.initials: G
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Yu
authors.forename: Peter
authors.initials: P
authors.lastname: Setlow
authors.forename: Yong-qing
authors.initials: YQ
authors.lastname: Li
mesh_terms: D001407:Bacillus / Q000502:physiology*; D008853:Microscopy / Q000379:methods*; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D052898:Optical Tweezers; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013171:Spores, Bacterial / Q000254:growth & development*