Resonance Raman spectral intensities per average bacterial cell have been measured quantitatively for Gram-negative Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes, as well as Gram-positive Bacillus subtilis and Staphylococcus epidermidis. Spectra have been obtained from cultures in the lag, log, and stationary growth phases excited in turn by 228.9, 244.0, and 248.2 nm light. Although Raman spectral peak positions (cm(-1)) excited by a given wavelength are very similar for all five bacterial species, the organisms are characterized by significantly different spectral intensity values. Intensity changes are associated with growth phase changes in all of the species as well. A comparison of measured with estimated average intensities has been made for spectra of log-phase E. coli. It is possible to compare measured intensities with intensities estimated for log-phase E. coli on the basis of the knowledge of its known average cellular molecular composition. A significant degree of hypochromism is observed in E. coli nucleic acid spectra. In contrast, strong average hyperchromism characterizes all aromatic amino acid peaks belonging to the same E. coli cells. Results suggest that knowledge of spectral intensity values will enhance significantly the capability to identify bacteria by means of their UV resonance Raman spectra.

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UV resonance Raman spectra of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis endospores have been excited at 222.7,230.7,242.5, and 251.1 nm, and spectra have been compared with those of vegetative cells. The resonance Raman spectra of aqueous solutions of dipicolinic acid and calcium dipicolinate have been measured at the same wavelengths. Spectra of endospores and their corresponding germinated spores show only modest differences when excited at 222, 231, and 251 nm. However, very substantial differences appear when excitation occurs at 242 nm. Difference spectra obtained at 242 nm by subtracting spectra of germinated spores of Bacillus cereus from spectra of their corresponding endospores are attributed almost entirely to dipicolinate. Vegetative cells and endospores show large spectral dissimilarities at all exciting wavelengths. These spectral differences, which vary strongly with exciting wavelength, appear to be the result of large differences in the amounts and composition of proteins and nucleic acids, especially ribosomal RNA. The very substantial resonance enhancement of Raman spectra has been obtained from aqueous solutions of pure dipicolinic acid and of sodium and calcium dipicolinate salts, as well as spores at the various exciting wavelengths. The strong enhancement of dipicolinate spectra in spores, however, was noted only with 242-nm excitation. Consequently, only with 242-nm light was it possible to selectively and sensitively excite and study calcium dipicolinate in spores. Resonance enhancement of the dipicolinate spectra with 242-nm excitation appears due primarily to resonance interactions with n-π* electronic transitions associated with the pyridine ring and/or the carboxylate group.