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

• Published in: Environmental microbiology 2009-01, Vol.11 (1), p.220-229
• Main Authors: Pumphrey, Graham M, Hanson, Buck T, Chandra, Subhash, Madsen, Eugene L
• Format: Article
• Language: English
• Subjects: Pseudomonas putida; Psuedomonas putida
• ISSN: 1462-2912; 1462-2920
• DOI: 10.1111/j.1462-2920.2008.01757.x

We demonstrate that dynamic secondary ion mass spectrometry (SIMS)-based ion microscopy can provide a means of measuring ¹³C assimilation into individual bacterial cells grown on ¹³C-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 ¹²C- and ¹³C-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 ¹²C⁻, ¹³C⁻, ²⁴(¹²C₂)⁻, ²⁵(¹²C¹³C)⁻, ²⁶(¹²C¹⁴N)⁻ and ²⁷(¹³C¹⁴N)⁻ ions respectively. We verified that ratios of signals taken from the same cells only changed minimally during a ~4.5 min period of primary O₂⁺ 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 ¹²C- to ¹³C-glucose: a standard curve was generated to predict ¹³C-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 ¹³C-labelled and unlabelled carbon sources to verify that differential ¹³C signals measured by SIMS were due to ¹³C assimilation into cell biomass. Finally, we made three key observations after applying SIMS ion microscopy to soil samples from a field experiment receiving ¹²C- or ¹³C-phenol: (i) cells enriched in ¹³C were heterogeneously distributed among soil populations; (ii) ¹³C-labelled cells were detected in soil that was dosed a single time with ¹³C-phenol; and (iii) in soil that received 12 doses of ¹³C-phenol, 27% of the cells in the total community were more than 90% ¹³C-labelled.