Identification of Live and Dead Bacteria: A Raman Spectroscopic Study

Abstract

Raman spectroscopy has been used to identify bacterial strains, bacterial components, such as protein and DNA bases, and the ratio of live to dead bacteria before and after exposure to ultraviolet (UV) radiation. New vibrational bands and changes in their intensity as a function of UV irradiation time have been recorded by high resolution Raman spectroscopy which made it possible to determine the mechanism of the UV inactivation of Escherichia coli (E. coli), Serratia marcescens (S. marcescens), and Micrococcus luteus (M. luteus) bacteria in saline solutions. We have also employed a novel, new, handheld spectrometer capable of recording, in situ, within minutes, the absorption, fluorescence, synchronous fluorescence, and Raman spectra of bacteria and other biological species and large molecules. INDEX TERMS Bacteria inactivation, optical spectroscopy, principal component analysis, Raman scattering.

Resonance Raman spectra for the in-situ identification of bacteria strains and their inactivation mechanism

“…This increase was seen in several different experimental trials and also with different baseline fits employed. Increase in the same wavenumber region has also been observed by us previously in our studies of Escherichia coli bacteria (22). Therefore this broad increase in the 1400 cm −1 region is inherent in the UV irradiated bacteria and is not a part of the baseline.…”

“…Analysis of PC1 (Fig. 7) shows a pronounced decrease in the Raman bands of carotenoids and a small increase in the 1400 cm −1 band, which we assign to protein photo-products (8,22) [21]. Plating and subsequent counting of Colony Forming Units (CFU), suggests that, the live bacteria population decreases roughly by 4 to 5 orders of magnitude within 5 minutes of UV irradiation (with UV intensity 5mW/cm 2 ).…”

“…The optical densities of the final bacterial suspensions were kept at ∼ 0.5 O.D, at 600nm, where the concentration is ∼ 10 8 cells/mL. The bench-top system used for recording the resonance Raman spectra and UV light irradiation has been described by us previously (22). Briefly, a Horiba Xplora Raman spectrometer with a 100X (0.90 N.A) microscope objective for excitation and collection of the spectra in an 180 • -backscattered geometry was used to record the Raman spectra of the bacteria being studied.…”

“…Therefore this broad increase in the 1400 cm −1 region is inherent in the UV irradiated bacteria and is not a part of the baseline. We had earlier assigned this increase to the denaturation of the bacterial proteins due to the formation of UV induced protein photoproducts based on the fact that UV irradiated pure proteins also showed a similar increase in this wavenumber region (22). Figures 4 and 5 show the ratio of the intensity of the prominent Raman bands, at 1525 cm −1 (C=C stretching vibrations of carotenoid polyene) to 1450 cm −1 lipid band and 1158 cm −1 (C-C stretching vibrations of carotenoid polyene) intensity to 1450 cm −1 lipid band intensity, respectively, as a function of UV irradiation dose.…”

“…Raman spectroscopy, however, has the disadvantage of requiring higher concentration of bacteria for accurate assignment of several relevant (protein, pigments, DNA) weak vibrational bands. For example, for Escherichia coli bacteria cells dispersed in water, a conventional Raman spectrum using visible excitation requires ∼ 10 12 cells/mL (21,22), whereas conventional fluorescence spectra exhibit a much stronger signal even at ∼ 10 7 cells/mL concentrations. This disadvantage, of Raman spectroscopy, can be, partly, overcome by sample concentration (e.g.…”