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The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coli, and Bacillus subtilishave been observed. Each organism exhibits a strong maximum in its emission spectrum at 330–340 nm when excited at 290 nm. Iodide quenching and denaturization experiments with 8 Murea provide strong evidence for the assignment of the 330–340-nm fluorescence to protein tryptophan. Most importantly, the decay of this bacterial protein-tryptophan fluorescence has been described by two exponential functions in all cases. The observation that characteristic protein-tryptophan fluorescence lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of bacteria. Direct application will most likely be found in combination with the measurement of other luminescence parameters.

Ultraviolet-excited (242 nm) resonance Raman spectra have been obtained for the first time for five types of bacteria: Escherichia coli, Pseudomonas fluorescens, Staphylococcus epidermidis, Bacillus subtilis, and Enterobacter cloacae. Detailed, highly reproducible spectra show substantial differences in both the intensities and the energies of peaks, which suggests that such spectra provide unique “fingerprints” reflecting the unique combinations of chemotaxonomic markers present in each type of organism. Many of the spectral features excited by 242-nm radiation probably arise from cellular RNA, DNA, and the amino acids tyrosine and tryptophan. Background fluorescence has been shown to be negligible.

Resonance Raman spectra of the gram-negative organism, Escherichia coli, have been obtained with 222.5-, 230.6-, and 251.0-nm excitation, and the results have been compared with those reported earlier for 242.4-nm excitation. Major changes in bacterial spectra have been observed with changes in exciting wavelength. The origins of the major peaks in each spectrum have been explained primarily in terms of contributions of nucleic acid bases and aromatic amino acids. As an aid in making assignments, spectra of aromatic amino acids, nucleosides, and mixtures of the two have been obtained at each wavelength used to excite bacterial spectra. Background fluorescence has been observed to be negligible below 251 nm. Selective excitation of bacterial nucleic acid and protein components has been done with ease. Results suggest that an extension of the exciting wavelength range to 190–220 nm will allow the selective excitation of additional cell components.

The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coli, and Bacillus subtilishave 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 widespread 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 Pseudomonas fluorescens, where one marker fluorophore, a pteridine, is produced in large amounts.

High-quality resonance Raman spectra in the visible region can be obtained from very small aggregates of chromobacteria, and very useful spectra can be obtained from individual cells. Individual cells in bacterial mixtures have been clearly identified by argon-laser-excited resonance Raman spectra taken with a commercially available Spex Micramate. Organisms present in the vicinity of the laser beam and in the beam itself can be observed and counted with ease by means of a television image obtained via a vidicon tube attached to a microscope. The method appears capable of identification at the species level.