Detection of Extracellular Phosphatase Activity of Heterotrophic Prokaryotes at the Single‐Cell Level by Flow Cytometry

Monitoring cell activity using substrates, which turn fluorescent due to biological activity, allows observing the presence and dynamics of sub‐populations, and provides a very valuable insight in ecological studies. The phosphatase substrate ELF97 phosphate (ELF‐P) is a useful tool to detect and quantify phosphatase activity (PA) of microorganisms at the single‐cell level. Most of the studies dealing with PA at the single‐cell level focus on autotrophic cells and only few concern heterotrophic prokaryotes (referred as bacteria in the text). While flow cytometry is a promising tool to assess the single‐cell analysis, only microscopy tools have been used until now to measure the ELF labeling associated with bacteria expressing PA. Therefore, we have developed a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF‐P hydrolysis, making possible the specific identification of bacteria showing PA using flow cytometry. Curr. Protoc. Cytom . 49:11.18.1‐11.18.8. © 2009 by John Wiley & Sons, Inc.