Raman spectroscopy was applied to study <i>Escherichia coli</i> and <i>Staphylococcus epidermidis</i> cells that were inactivated by different chemicals and stress conditions including starvation and high temperature. <i>E. coli</i> cells exposed to starvation conditions over several days lost viability at the same rate that spectral bands assigned to DNA and RNA bases decreased in intensity. Band intensities correlate with standard plate counts with <i>R<sup>2</sup></i> = 0.99 and <i>R<sup>2</sup></i> = 0.97, respectively. Principal components analysis and discriminant analysis multivariate statistical techniques were used to evaluate the spectral data collected. Significant changes were observed in the spectra of treated cells in comparison with their respective controls (samples without treatment). As a result, there was a significant differentiation between viable and non-viable cells (treated and non-treated cells) in the first and second principal component plots for all the treatments. Discriminant analysis was used along with PCA to estimate a classification rate based on viability status of the cells. Non-viable cells were differentiated from viable cells with classification rates that ranged between 60 and 90% for specific treatments (i.e., EDTA-treated cells versus control cells). The classification rate obtained considering all the treatments (non-viable cells) and controls (viable cells) at the same time for each of the species studied was 86%. The classification rate based on species differentiation when all the spectra (viable and non-viable) were used was 87%. These results suggest that Raman spectroscopy is a powerful tool that can be used to evaluate viability and to study metabolic changes in microorganisms. It is a robust method for bacterial identification even when high spectral variations are introduced.

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