Using Stable Isotope Probing and Raman Microspectroscopy To Measure Growth Rates of Heterotrophic Bacteria

The suitability of stable isotope probing (SIP) and Raman microspectroscopy to measure growth rates of heterotrophic bacteria at the single-cell level was evaluated. Label assimilation into Escherichia coli biomass during growth on a complex 13C-labeled carbon source was monitored in time course experiments. 13C incorporation into various biomolecules was measured by spectral "red shifts"of Raman-scattered emissions. The 13C- and 12C-isotopologues of the amino acid phenylalanine (Phe) proved to be quantitatively accurate reporter molecules of cellular isotopic fractional abundances (fcell). Values of fcell determined by Raman microspectroscopy and independently by isotope ratio mass spectrometry (IRMS) over a range of isotopic enrichments were statistically indistinguishable. Progressive labeling of Phe in E. coli cells among a range of 13C/12C organic substrate admixtures occurred predictably through time. The relative isotopologue abundances of Phe determined by Raman spectral analysis enabled the accurate calculation of bacterial growth rates as confirmed independently by optical density (OD) measurements. The results demonstrate that combining SIP and Raman microspectroscopy can be a powerful tool for studying bacterial growth at the single-cell level on defined or complex organic 13C carbon sources, even in mixed microbial assemblages.