Fig 1.
(A) The 500-Year Microbiology Experiment and its components. (B) Glass vials containing 100 μL of B. subtilis spore stock solution (106 CFU/mL) and then dried down on silicon bead desiccators before being sealed as described in Methods. (C) Silica beads to maintain desiccation.
Table 1.
Conditions for storage of air-dried B. subtilis spores*.
Table 2.
Conditions for liquid storage of B. subtilis spores*.
Fig 2.
Live cell microscopy of germinating B. subtilis spores recovered from baseline samples in 500-year storage conditions.
Images of three time points of germination are shown: 0, 2.5 and 4 h (see S1 Movie for an entire image sequence at 5 sec intervals). Note, that time point 0 h marks the beginning of the imaging. Activation of spores started 1–2 minutes before by adding a layer of LB-agar on top of the spores (see Materials & Methods section). A subpopulation of spores (~ 17%) was not capable of germination (arrows). Non-germinating spores appeared rather grey in phase-contrast without the bright core and darker ring-like boundary which is typical for dormant spores capable of germination (see S2 Fig). Scale bar = 5 μm.
Table 3.
Resistance of B. subtilis baseline spores of the 500-year experiment to various agents*.
Fig 3.
Spore survival during storage in space-like vacuum (10−7 Pa).
Dry spores were stored at 10−7 Pa and spore survival was determined as described in Methods. Error bars represent standard deviation. Significance by ANOVA between different incubation times is denoted by (*) with p<0.001.
Table 4.
LD90 (90% spore inactivation) ranges for storage under different conditions*.
Fig 4.
Spore survival in various NaCl solutions.
Spores, 108 per sample, were stored in solutions with various NaCl concentrations, and spore survival was measured as described in Methods. Black squares denote spores in water (0 M NaCl); blue diamonds denote spores in 1.2 M NaCl; and red circles denote spores in 3.6 M NaCl. Error bars signify the standard deviation.