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.