Spore Germination and DPA Quantification

Germination was monitored using a Spectramax M3 plate reader (Molecular Devices, Sunnyvale, CA). 5 μL of OD₆₀₀ = 100 spores were added to 95 μl germination buffer consisting of a final concentration 1x HEPES, 30 mM glycine, 10 mM TA and the OD₆₀₀ was monitored for 1 hour at 37°C. To assay total DPA content, 1 x 10⁸ spores in 20 μL, were boiled at 95°C for 20 minutes. 5 μL (an equivalent of 2.5 x 10⁷ spores) of the solution was added to 95 μL of 1X HEPES buffer with 250 μM TbCl₃ and analyzed by excitation at 275 nm and emission at 545 nm with a 420 nm cutoff [54 (link),55 (link),59 (link),66 (link),79 (link)].