Figure 1.
Raman spectra of individual spores. Raman spectra of individual G. stearothermophilus (A), B. subtilis (B), and B. cereus spores (C), measured at 25°C (curve a), 65°C (curve b) and 95°C (curve c), respectively.
Curve d in Fig. 1(C) is the Raman spectrum of single B. cereus spores that had lost their CaDPA at 95°C. All the spectra were averages from 30 individual spores determined as described in Methods. The dotted lines are the protein bands of amide I (1653/1667 cm−1) and amide III (1253 cm−1), respectively.
Table 1.
CaDPA level in individual G. stearothermophilus and Bacillus spores*.
Figure 2.
Dynamics of nutrient germination of an optically trapped individual G. stearothermophilus spore.
A heat activated (30 min, 100°C) spore was germinated at 65°C with 0.1 mM L-valine in 10 mM sodium phosphate buffer (pH 8.0), and the spore was monitored by Raman spectroscopy and DIC microscopy as described in Methods. Time-lapse Raman spectra of the trapped spores after the addition of L-valine were shown in (A). The indicated peaks at 661, 825, 1,017, 1,395 and 1,575 cm−1 are the CaDPA bands. Normalized intensities of the CaDPA band at 1017−1 (○) and DIC images (□) as the function of incubation time were shown in (B). The CaDPA band intensities and DIC image intensities were normalized to their initial values right after the addition of L-valine, and the DIC image intensity at 25 min was normalized to 0. The interval between Raman spectrum acquisitions was 30 s, and the interval between DIC image acquisitions was 15 s. The inserts in Fig. 2(B) are the time-lapse DIC images of the trapped spore with a scale bar of 2 µm. The DIC image of a single spore appears as two bright spots in DIC microscopy.
Figure 3.
Effects of different activation methods on germination of multiple individual G. stearothermophilus spores.
Spores were activated by various methods, and germinated at 65°C with 1.0 mM L-valine and 10 mM sodium phosphate buffer (pH 8.0), and germination of individual spores was monitored by DIC microscopy as described in Methods. Germination of ≥248 individual spores (Table 2) that were activated in 0.2 M sodium nitrite (pH 8.0) at 30°C for 17 h (•), in water at 30°C for 120 h (▴), in water at 100°C for 30 min (▪), or without activation (*) was shown in (a). Kinetics of germination of ten individual spores without activation (b); activated at 30°C for 120 h (c), and activated in 0.2 M sodium nitrite (pH 8.0) for 17 h (d) was given in (b-d).
Table 2.
Effect of activation methods on G. stearothermophilus spore germination*.
Figure 4.
L-Valine germination of multiple individual G. stearothermophilus spores.
Heat activated spores (30 min, 100°C) were germinated at various temperatures with 1 mM L-valine in 10 mM sodium phosphate buffer (pH 8.0), and germination of individual spores was monitored by DIC microscopy as described in Methods. Germination of ≥264 individual spores at 65°C(▪), 55°C (•), or 45°C (▴) was shown in (a). Kinetics of germination of ten individual spores at 65°C (b), 55°C (c), or 45°C (d) was given in (b–d).
Figure 5.
Germination of multiple individual G. stearothermophilus spores with AGFK at different temperatures.
Heat activated (30 min, 100°C) spores were germinated at various temperatures with AGFK and 10 mM sodium phosphate buffer (pH 8.0), and germination of individual spores was monitored by DIC microscopy as described in Methods. Germination of ≥220 individual spores at 65°C (▴), 55°C (•), or 45°C (▪) was shown in (a). Kinetics of germination of ten individual spores at 65°C (b), 55°C (c), or 45°C (d) was given in (b–d).
Table 3.
Mean values and standard deviations of Tlag, Trelease, ΔTrelease, Tlys, and ΔTlys values for individual germinating G. stearothermophilus spores*.
Figure 6.
Germination of multiple individual G. stearothermophilus spores with CaDPA.
Unactivated spores were germinated with CaDPA at various temperatures, and germination of individual spores was followed by DIC microscopy as described in Methods. Germination at 65°C (▪) or 25°C (•), with germination of ≥380 individual spores examined was shown in (a). Kinetics of germination of ten individual spores at 65°C (b) or25°C (c) was given in (b, c).
Figure 7.
Germination of multiple individual G. stearothermophilus spores with dodecylamine.
Unactivated spores were germinated at various temperatures with 1(pH 8.0), and germination of ≥470 individual spores was monitored by DIC microscopy. Germination at 65°C (▪), 55°C (•) and 45°C (▴) was shown in (a). Kinetics of germination of ten individual spores at 65°C (b) or 55°C (c) was given in (b, c).
Figure 8.
Germination of multiple individual decoated G. stearothermophilus spores.
Heat activated (30 min, 100°C (a,b), or unactivated spores (c,d) were germinated at 65°C with 1 mM L-valine (a); 1 mM AGFK (b); 60 mM CaDPA (c); and 1 mM dodecylamine (d), in 10 mM sodium phosphate buffer (pH 8.0), and germination of individual spores was monitored by DIC microscopy as described in Methods. The insets in the various panels show the percentages of spore germination when ≥224 individual spores (Table 2) were monitored.