Effect of Cultural Conditions on Deep UV Resonance Raman Spectra of Bacteria

Abstract

Bacteria grown on trypticase soy agar (TSA), trypticase soy broth (TSB), and Davis minimal media, and harvested at times ranging from 4.5 to 48 h have been excited at 242.54 and 222.65 nm for the purpose of generating resonance Raman spectra. When excitation with 242.54-nm light occurs, simple spectra of tyrosine and tryptophan and various nucleic acids are observed. Large changes in the relative intensities of major nucleic acid peaks at 1485 and 1575 cm−1, on the one hand, as compared to a prominent protein tyrosine + tryptophan peak at 1616 cm−1, on the other, have been attributed to very large variations in the RNA content of bacterial cells from culture to culture. The spectral changes are observed whenever differences in growth rates or variations in cultural media result in substantial changes in the amount of ribosomal RNA. In spite of very large cultural effects on peak intensities it has been possible to obtain bacterial G + C/A + T ratios from these spectra. Specifically, the ratio of the intensity of the C (1530 cm−1) peak to the intensity of the A + G peak (1485 cm−1) when plotted against the known molar percent G + C of the corresponding bacterial DNA produces a straight line. Plots have been shown to be very nearly growth-time and media independent for fourteen different types of bacteria, which range in DNA G + C content from 32 to 66%. Spectra obtained with 222.65-nm light, in contrast with spectra obtained with 242.54-nm excitation, have been found to be nearly growth-rate and media independent. The excitation wavelength, 222.65 nm, appears to be the best yet found for use in rapid Raman identification of bacteria. All strong peaks which have been assigned have been attributed to protein modes. Relative intensities of 1556-cm−1 tryptophan and 1616-cm−1 tryptophan + tyrosine bands have been found to be strongly correlated with bacterial Gram type and nearly independent of cultural media or stage of growth.