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Dynamic secondary ion mass spectrometry imaging of microbial populations utilizing 13 C‐labelled substrates in pure culture and in soil
Author(s) -
Pumphrey Graham M.,
Hanson Buck T.,
Chandra Subhash,
Madsen Eugene L.
Publication year - 2009
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2008.01757.x
Subject(s) - pseudomonas putida , secondary ion mass spectrometry , ion , analytical chemistry (journal) , mass spectrometry , biology , pseudomonas , pseudomonadaceae , bacteria , chromatography , chemistry , genetics , organic chemistry
Summary We demonstrate that dynamic secondary ion mass spectrometry (SIMS)‐based ion microscopy can provide a means of measuring 13 C assimilation into individual bacterial cells grown on 13 C‐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 12 C‐ and 13 C‐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 12 C – , 13 C – , 24 ( 12 C 2 ) – , 25 ( 12 C 13 C) – , 26 ( 12 C 14 N) – and 27 ( 13 C 14 N) – ions respectively. We verified that ratios of signals taken from the same cells only changed minimally during a ∼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 12 C‐ to 13 C‐glucose: a standard curve was generated to predict 13 C‐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 13 C‐labelled and unlabelled carbon sources to verify that differential 13 C signals measured by SIMS were due to 13 C assimilation into cell biomass. Finally, we made three key observations after applying SIMS ion microscopy to soil samples from a field experiment receiving 12 C‐ or 13 C‐phenol: (i) cells enriched in 13 C were heterogeneously distributed among soil populations; (ii) 13 C‐labelled cells were detected in soil that was dosed a single time with 13 C‐phenol; and (iii) in soil that received 12 doses of 13 C‐phenol, 27% of the cells in the total community were more than 90% 13 C‐labelled.