This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. 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 information leads to precise correlations between various germination events, and takes 1-2 h to complete. 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.

Dormant bacterial spores do not take up and bind nucleic acid dyes in the spore core but readily take up such dyes when they are fully germinated. We present a methodology that combines fluorescence microscopy, phase contrast microscopy, and laser tweezers Raman spectroscopy to monitor the kinetics of uptake of the nucleic acid dye SYTO 16 during germination of individual Bacillus cereus and Bacillus subtilis spores. The level of dye bound to nucleic acids of individual spores was measured by fluorescence emission, while changes in spore refractility and the level of the 1:1 chelate of dipicolinic acid and Ca(2+) (CaDPA) were monitored by phase contrast microscopy and Raman spectroscopy, respectively. The results obtained include (1) during nutrient germination, SYTO 16 began to enter the spore core and bind to nucleic acids just when spores had released all CaDPA and continued until hydrolysis of spores' peptidoglycan cortex was complete; (2) during germination with exogenous CaDPA, rapid SYTO 16 uptake began only 2-7 min after complete release of endogenous CaDPA for both B. cereus and B. subtilis spores; (3) the rate but not the timing of dye uptake and the maximum level of dye bound to nucleic acid were increased during nutrient germination of B. subtilis spores lacking ~75% of the DNA binding proteins that normally saturate dormant spore DNA; (4) SYTO 16-DNA binding was not observed during nutrient germination of B. subtilis spores lacking the protease that degrades spores' DNA binding proteins, even after cortex hydrolysis; (5) SYTO 16 uptake by germinating B. subtilis spores lacking the cortex-lytic enzyme (CLE) CwlJ was low, again even after cortex hydrolysis, although SYTO 16 uptake by germinating spores lacking the other redundant CLE SleB was even higher than in germinating wild-type spores; and (6) there was no SYTO 16 uptake by germinating spores that lacked both CwlJ and SleB, even after CaDPA release. These results suggest that during spore germination SYTO 16 uptake is minimal until CaDPA has been released and DNA binding proteins have been degraded and further that CLEs' degradation of the spore cortex plays a crucial role in uptake of this dye.

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