volume of 20 mM terbium chloride in Tris buffer (pH 7.5). Fluorescence measurements were done in a spectrofluorometer (Varioskan Flash, Thermo Electron Corporation, Nepean, Canada) with
excitation and emission wavelengths of 270 and 545 nm, respectively. Results were calculated as % DPA release relative to control
samples prepared from the same endospore suspension. Fluorescence was recorded as relative fluorescence units (RFU).
Terbium-DPA fluorescence assays were done to establish
a relationship between endospore density and % DPA release
following lethal thermal treatments of endospores. Suspensions of
C. beijerinckii endospores, standardized to OD600 values of 0.25, 0.5,
1.0, and 2.0, were heated at 120
C for 60 min. Increasing OD600
values resulted in a corresponding increase of the amount of DPA
released from the endospores (data not shown). Treatment of
endospores at 120
C caused a near complete DPA release in under
a minute for all three Clostridium spp. (data not shown).