Dynamic secondary ion mass spectrometry imaging of microbial populations utilizing C-13-labelled substrates in pure culture and in soil

We demonstrate that dynamic secondary ion mass spectrometry (SIMS)-based ion microscopy can provide a means of measuring C-13 assimilation into individual bacterial cells grown on C-13-labelled organic compounds in the laboratory and in field soil. We grew pure cultures of Pseudomonas putida NCIB 9816-4 in minimal media with known mixtures of C-12- and C-13-glucose and analysed individual cells via SIMS imaging. Individual cells yielded signals of masses 12, 13, 24, 25, 26 and 27 as negative secondary ions indicating the presence of C-12(-), C-13(-), (24)(C-12(2))(-), (25)((CC)-C-12-C-13)(-), (26)((CN)-C-12-N-14)(-) and (27)((CN)-C-13-N-14)(-) ions respectively. We verified that ratios of signals taken from the same cells only changed minimally during a similar to 4.5 min period of primary O-2(+) beam sputtering by the dynamic SIMS instrument in microscope detection mode. There was a clear relationship between mass 27 and mass 26 signals in Psuedomonas putida cells grown in media containing varying proportions of C-12- to C-13-glucose: a standard curve was generated to predict C-13-enrichment in unknown samples. We then used two strains of Pseudomonas putida able to grow on either all or only a part of a mixture of C-13-labelled and unlabelled carbon sources to verify that differential C-13 signals measured by SIMS were due to C-13 assimilation into cell biomass. Finally, we made three key observations after applying SIMS ion microscopy to soil samples from a field experiment receiving C-12- or C-13-phenol: (i) cells enriched in C-13 were heterogeneously distributed among soil populations; (ii) C-13-labelled cells were detected in soil that was dosed a single time with C-13-phenol; and (iii) in soil that received 12 doses of C-13-phenol, 27% of the cells in the total community were more than 90% C-13-labelled.