Detection of extracellular phosphatase activity at the single-cell level by enzyme-labeled fluorescence and flow cytometry: the importance of time kinetics in ELFA labeling.

ELF97 phosphate (ELF-P) is a useful compound for assessing the phosphorus-related status of planktonic aquatic populations. The technique has been successfully applied to phytoplankton and more recently to heterotrophic prokaryotes in both freshwater and marine samples. We have used a recently developed protocol that enables the detection by flow cytometry of ELF alcohol (ELFA), the product of ELF-P hydrolysis. This protocol allows for identification of the fraction of cells able to express phosphatase activity (i.e., ELFA-labeled). This protocol is also very valuable in the study of time kinetics in this ELFA-labeling. The percentage of ELFA-labeled cells, the relative median ELFA fluorescence per cell, and the absolute ELFA fluorescence were determined in both freshwater (lake) and marine samples. The incubation time necessary to reach a stable percentage of active cells with maximal fluorescence intensity varied widely among samples. We highlight very subtle but important problems of discrimination between active and nonactive cells and of estimation of per-cell activity and we underline the importance of studying time kinetics of ELFA-labeling to determine the appropriate incubation time and thus making sample comparisons more relevant. Working on time kinetics of ELFA-labeling is promising for phosphomonoester hydrolysis rate determination at single cell level.