Real-time detection of kinetic germination and heterogeneity of single Bacillus spores by laser tweezers Raman spectroscopy

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.