Real-Time Detection of Kinetic Germination and Heterogeneity of Single Bacillus Spores by Laser Tweezers Raman Spectroscopy

Abstract

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.

Methods and Applications of Raman Microspectroscopy to Single-Cell Analysis

“…A number of studies have shown that Raman scattering-based methods allow for the assessment of the type of contamination, as well as the degree of contamination in such applications. [79][80][81][82][83][84] In early work, Choo-Smith et al 85 86 used Raman spectroscopy to quantify filtered waterborne pathogenic microbial contamination, and Pasteris et al 87 used Raman spectroscopy to identify sulfur inclusions in sulfur-oxidizing marine bacteria. Recently, Palchaudhuri et al 88 investigated the uptake and metabolism of the five-carbon sugar alcohol xylitol by gram-positive viridian group Streptococcus and two gram-negative Escherichia coli strains.…”

Quantitative Analysis of Spatial-Temporal Correlations during Germination of Spores of Bacillus Species

“…While Ca-DPA solubility is ϳ70 mM, the Ca-DPA concentration in total dormant spore water would be ϳ90 mM if all were soluble, assuming the weight of a B. subtilis spore is 6 ϫ 10 Ϫ13 g, contains 10% dry weight as DPA, and is ϳ70% water (17,28). However, full release of Ca-DPA from the core during germination takes 1 to 3 min for an individual spore (4,13,22,35), indicating that there is ample time for tremendous dilution of this released Ca-DPA well before an adjacent spore is encountered. Consequently, it is perhaps not unexpected that Ca-DPA released from one spore is not especially effective in triggering the germination of an adjacent spore.…”

“…Unfortunately, the rates of germination of individual spores are extremely heterogeneous due to the variations in t lag (4,11,13,35). Some of the factors that influence t lag values have recently been elucidated (35), although there could well be additional important factors.…”

“…A key early step in germination is the release of the spores' large pool (ϳ20% of the dry weight of spores' central region or core) of dipicolinic acid (DPA) that is chelated to divalent metal ions, predominantly Ca 2ϩ (Ca-DPA) (12,25,27). Recent studies with individual spores have shown that once rapid release of Ca-DPA during germination has begun, it is completed in only a few minutes (4,13,22,27,35). However, there are generally long lag times (t lag ) between the time of addition of a nutrient germinant to the initiation of rapid Ca-DPA release for individual spores, and t lag values vary greatly from spore to spore even in genetically identical populations germinating under the same conditions (4,35).…”

“…Recent studies with individual spores have shown that once rapid release of Ca-DPA during germination has begun, it is completed in only a few minutes (4,13,22,27,35). However, there are generally long lag times (t lag ) between the time of addition of a nutrient germinant to the initiation of rapid Ca-DPA release for individual spores, and t lag values vary greatly from spore to spore even in genetically identical populations germinating under the same conditions (4,35). In contrast, the time needed for release of ϳ90% of a spore's Ca-DPA during germination is relatively constant at several minutes.…”