Carbon-isotopic analysis of microbial cells sorted by flow cytometry.

Abstract

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 δ13 C in whole cells with precision better than 0.2‰ for samples containing as little as 25ng of carbon. When losses associated with sample handling are taken into account, isotopic analyses require sorting roughly 104 eukaryotic or 107 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.