Improved methodology to measure taxon-specific phosphate uptake in live and unfiltered samples
Limnol. Oceanogr. Methods 9:443-453 (2011) | DOI: 10.4319/lom.2011.9.443
ABSTRACT: Microorganisms play a major role in the marine phosphate biogeochemical cycle but the relative contribution of picoplanktonic groups is not well understood. Previous studies have shown that combining uptake measurements of radiolabeled dissolved inorganic phosphate (Pi) substrate with cell sorting by flow cytometry is a powerful tool for the assessment of Pi fluxes at the cell-specific level. Nevertheless, using 33P to trace Pi uptake, we show that treatments involving fixation and filtration of the sorted groups (i.e., heterotrophic prokaryotes, Synechococcus and piconanophytoeukaryotes) induce leakage of radioactive Pi (up to 50% of the signal), resulting in a sizeable underestimation of the taxon-specific Pi uptake. We suggest an alternative protocol, which significantly reduces this bias. Using this optimized protocol, the samples were treated with an excess of nonradioactive Pi to stop the incubation and sorted fractions were directly collected in microtubes for radioactivity counting, avoiding signal loss due to filtration. Sorted groups were strongly and differently impacted by fixation (0.5% PFA), with the exception of Synechococcus cells, which showed once a 10% lower signal in samples treated with the previously used protocols compared with samples treated with ours. Based on the integrity of the live sorted cells, our improved protocol provides reproducible and accurate estimations of the taxon-specific Pi uptake (≤ 11% variation on cellular uptake rates, sd/average, n = 69). It was successfully applied to P-depleted oligotrophic seawater samples from the Mediterranean and will allow a comparison of taxon-specific uptake rates between sites.