Germination is the process by which a dormant spore returns to its vegetative state when exposed to suitable conditions. We report on the real-time detection of kinetic germination and heterogeneity of single Bacillus thuringiensis spores in an aqueous solution by monitoring the calcium dipicolinate (CaDPA) biomarker with laser tweezers Raman spectroscopy (LTRS). A single B. thuringiensis spore was optically trapped in a focused laser beam, and its Raman spectra were recorded sequentially in time after exposure to a nutrient-rich medium, so that the CaDPA amount inside the trapped spore was monitored during the dynamic germination process. The CaDPA content in an individual spore was observed to remain almost constant in the first period and then decrease very rapidly due to its release into the medium (within approximately 2 min). The time-to-germination (t(germ)), defined as the time required for the CaDPA band intensity to decrease to the midpoint from its initial value, was found to be stochastic for individual spores with a typical value of approximately 30 min under the experimental conditions. The distribution of the time-to-germination was measured from a time lapse measurement of a population of spores. The results demonstrated that LTRS can be used to noninvasively detect the kinetic germination process at the single-cell level and explore cellular heterogeneity.

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 approximately 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.

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