Characterization of Bacterial Spore Germination Using Integrated Phase Contrast Microscopy, Raman Spectroscopy, and Optical Tweezers

We present a methodology that combines external phase contrast microscopy, Raman spectroscopy, and optical tweezers to monitor a variety of changes during the germination of single Bacillus cereus spores in both nutrient (L-alanine) and non-nutrient (Ca-dipicolinic acid (DPA)) germinants with a temporal resolution of similar to 2 s. Phase contrast microscopy assesses changes in refractility of individual spores during germination, while Raman spectroscopy gives information on changes in spore-specific molecules. The results obtained include (1) the brightness of the phase contrast image of an individual dormant spore is proportional to the level of CaDPA in that spore; (2) the end of the first Stage of germination, revealed as the end of the rapid drop in spore refractility by phase contrast microscopy, precisely corresponds to the completion of the release of CaDPA as revealed by Raman spectroscopy; and (3) the correspondence between the rapid drop in spore refractility and complete CaDPA release was observed not only for spores germinating in the well-controlled environment of an optical trap but also for spores germinating when adhered on a microscope coverslip. Using this latter method, we also simultaneously characterized the distribution of the time-to-complete-CaDPA release (T-release) of hundreds of individual B. cereus spores germinating with both saturating and subsaturating concentrations of L-alanine and with CaDPA.