FIG. 1.

Equilibrium density gradient centrifugation of spore populations with various percentages of spores killed by moist heat. Spores of strain PS533 (wild type) were either untreated (tube 1) or treated for increasing times at ∼89°C (40, 80, and 120 min for tubes 2 to 4, respectively), and viability was determined; spores were run on Nycodenz step gradients, and the spores in the upper (U) or lower (L) bands were isolated. The viability of the spores was determined as the number of CFU/0.5 ml of spores at an OD₆₀₀ of 1, as described in Materials Methods. The values for the viability of untreated spores before centrifugation or from the lower band in tube 1 were each set at 100%, and the absolute viabilities for these two samples were almost identical. Values for the viability of spores from upper and lower bands in tubes 2 to 4 are expressed relative to the value for the lower band from untreated spores in tube 1. Based on the OD₆₀₀ of spores recovered from the upper and lower bands, the upper band comprised 4%, 20%, and 60% of total spores in tubes 2, 3, and 4, respectively. Since the upper band had lost DPA and this decreases the OD₆₀₀ of spore suspensions ∼25% (28), the ratios of the amounts of spores in the upper to the lower bands are 0.05, 0.3, and 3 in tubes 2 to 4, respectively. This experiment was repeated three times with essentially identical findings.