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Carbon-isotopic analysis of microbial cells sorted by flow cytometry

Eek, K. M. and Sessions, A. L. and Lies, D. P. (2007) Carbon-isotopic analysis of microbial cells sorted by flow cytometry. Geobiology, 5 (1). pp. 85-95. ISSN 1472-4677. doi:10.1111/j.1472-4669.2006.00094.x.

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One of the outstanding current problems in both geobiology and environmental microbiology is the quantitative analysis of in situ microbial metabolic activities. Techniques capable of such analysis would have wide application, from quantifying natural rates of biogeochemical cycling to identifying the metabolic activity of uncultured organisms. We describe here a method that represents one step towards that goal, namely the high-precision measurement of ^(13)C in specific populations of microbial cells that are purified by fluorescence-activated cell sorting (FACS). Sorted cells are concentrated on a Teflon membrane filter, and their ^(13)C content is measured by coupling an isotope ratio mass spectrometer (IRMS) with a home-built spooling wire microcombustion (SWiM) apparatus. The combined instrumentation provides measurements of δ^(13)C in whole cells with precision better than 0.2‰ for samples containing as little as 25 ng of carbon. When losses associated with sample handling are taken into account, isotopic analyses require sorting roughly 10^4 eukaryotic or 10^7 bacterial cells per sample. Coupled with ^(13)C-labelled substrate additions, this approach has the potential to directly quantify uptake of metabolites in specific populations of sorted cells. The high precision afforded by SWiM-IRMS also permits useful studies of natural abundance variations in ^(13)C. The approach is equally applicable to specific populations of cells sorted from multicellular organisms.

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Sessions, A. L.0000-0001-6120-2763
Additional Information:© 2006 The Authors. Journal compilation © 2006 Blackwell Publishing Ltd. Received 04 August 2006; accepted 09 November 2006. Article first published online: 21 Dec. 2006. We wish to express our gratitude to Annelie Pernthaler for assisting in the development of cell concentrating methods, Sky Rashby for growing cyanobacterial cultures, Dane Mohl for providing S. cervisae strains, Shelley Diamond for assistance with flow cytometry, Chris Baumgartner, Rick Paniagua and Rick Gerhart for assisting with construction of the SWiM device. We also thank Victoria Orphan and Dianne Newman for sharing ideas and resources. KME and ALS are supported by NSF grant OCE-0321339 and by the Beckman Institute at California Institute of Technology.
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Caltech Beckman InstituteUNSPECIFIED
Issue or Number:1
Record Number:CaltechAUTHORS:20130610-111937609
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Official Citation:EEK, K. M., SESSIONS, A. L. and LIES, D. P. (2007), Carbon-isotopic analysis of microbial cells sorted by flow cytometry. Geobiology, 5: 85–95. doi: 10.1111/j.1472-4669.2006.00094.x
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:38878
Deposited By: Tony Diaz
Deposited On:24 Jun 2013 22:20
Last Modified:09 Nov 2021 23:40

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