Commonly used biological indicators for assessing the efficiency of decontamination procedures include the spores of Bacillus atrophaeus (BA) and Geobacillus stearothermophilus (GS). BA and GS spores emit tryptophan-like fluorescence around 320 nm when excited by 280 nm UV light. This fluorescence signal is highly dependent on vaporised hydrogen peroxide (VHP) concentration around the spores, since VHP quenches the fluorescence. In this study we investigated the precise influence of VHP concentration on the autofluorescence properties of BA and GS spores. For both types of spores, the fluorescence signal intensity was found to fall faster and to a relatively lower level when higher VHP concentration was applied. The shape of the signal fall-off as a function of time was found to be well approximated by biexponential decay functions with similar time-constants for BA and GS spores, finally resulting in the equilibrium (or plateau/residual) fluorescence intensity levels that were very different for these samples. The reasons for the fluorescence signal fall-off were investigated by spectral fluorescence signatures (SFS) of the spores. The SFS measurements of the spores during VHP decontamination revealed that the spectral maximum of tryptophan-like (Trp-like) fluorescence changes towards a smaller Stokes shift and has the intensity fall-off due to quenching and oxidation under VHP influence.
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