![]() ![]() Observation of a single-beam gradient force optical trap for dielectric particles. Monitoring the kinetics of Bacillus subtilis endospore germination via surface-enhanced Raman scattering spectroscopy. Monitoring DPA release from a single germinating Bacillus subtilis endospore via surface-enhanced Raman scattering microscopy. ![]() Flow cytometric assessment of Bacillus spore response to high pressure and heat. Mathys, A., Chapman, B., Bull, M., Heinz, V. A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies. Factors influencing germination of Bacillus subtilis spores via activation of nutrient receptors by high pressure. Assessment of bacterial endospore viability with fluorescent dyes. Analysis of nucleoid morphology during germination and outgrowth of spores of Bacillus species. In vitro high-resolution structural dynamics of single germinating bacterial spores. Plomp, M., Leighton, T.J., Wheeler, K.E., Hill, H.D. Imaging and analysis of Bacillus anthracis spore germination. Dipicolinic acid (DPA) assay revisited and appraised for spore detection. ![]() Real time monitoring of Bacillus subtilis endospore components by attenuated total reflection Fourier transform infrared spectroscopy during germination. Study of calcium dipicolinate release during bacterial spore germination by using a new, sensitive assay for dipicolinate. Release of small molecules during germination of spores of Bacillus species. & Setlow, P.) 43–63 (American Society for Microbiology, 1989). In Regulation of Prokaryotic Development (eds. Spores of Bacillus subtilis: their resistance to radiation, heat and chemicals. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J. In Food Microbiology: Fundamentals And Frontiers 3rd edn. 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. This information leads to precise correlations between various germination events, and takes 1–2 h to complete. 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 protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. ![]()
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