Selection of germination mutants (correspondence, Junichi Sekiguchi)

      B. subtilis 168 and disruptants were cultured on LB plates for 12 h at 37 C and then cells were inoculated into 5 ml DSM (Schaeffer) medium followed by incubation for 4 h at 37 C. After centrifugation of the preculture, cells were cultured in 50 ml DSM medium for 52-60 h (until T48) at 37 C. After centrifugation of the culture, a mixture of free spores and sporangia were suspended in 10 ml deionized water, and then washed by centrifugation until all the cell debris and vegetative cells have been removed. For the routine experiments, the "spore washing" was repeated for 2 weeks (one washing a day). The purified spores (0.3 A580) were heat-activated at 80 C for 20 min and then diluted with a 10 mM Tris-HCl buffer (pH 8.4). Germination was initiated by the addition of L-alanine to a final concentration of 10 mM or AGFK (L-asparagine, D-glucose, D-fructose and KCl) to final concentrations of 10, 1, 1 and 10 mM, respectively. At appropriate times (0, 30, 60, 90 and 120 min for the routine experiments), the A580 of the mixture was measured.