The intrinsic steady-state fluorescence and fluorescence decay of <i>Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coli,</i> and <i>Bacillus subtilis</i> have been observed. Excitation spectra were obtained while emission at 430, 455, 487 and 514 nm was being monitored. Emission spectra were obtained with the use of excitation wavelengths of 340, 365, 405, 430 and 460 nm. Fluorescence lifetimes were measured at 430, 487, and 514 nm while selective excitation was caused at 340, 405, and 430 nm. The complex nature of the excitation and emission spectra reflects the presence of a number of different fluorophores. Attempts have been made to describe portions of the bacterial fluorescence in terms of the measured fluorescence properties including lifetimes of molecular components known for their wide-spread occurrence in bacteria and their relatively high quantum yields. Candidate fluorophores which have been considered include the pteridines, the structurally related flavins, and the pyridine coenzymes. The observation that characteristic sets of lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of bacteria. Results are especially definitive in cases such as <i>Pseudomonas fluorescens,</i> where one marker fluorophore, a pteridine, is produced in large amounts.

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