A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry

Area of Science:

• Microbiology
• Environmental Science
• Biotechnology

Background:

• Phosphatase activity is crucial for phytoplankton and heterotrophic bacteria, especially in phosphorus-limited environments.
• Previous methods using ELF97-phosphate (ELF-P) were limited to epifluorescence microscopy for marine bacteria.
• A need exists for a more efficient and quantitative method to assess bacterial phosphatase activity in natural samples.

Purpose of the Study:

• To develop and validate a novel flow cytometry protocol for detecting ELF alcohol (ELFA), the product of ELF-P hydrolysis.
• To enable the detection and quantification of phosphatase-positive heterotrophic bacteria in natural aquatic samples.
• To establish a method for sample processing, storage, and analysis compatible with flow cytometry.

Main Methods:

• Disaggregation of bacterial clumps using Tween 80 and concentration via centrifugation.
• Incubation with ELF97-phosphate (ELF-P) to detect phosphatase activity, yielding ELF alcohol (ELFA).
• Flow cytometry analysis to detect ELFA precipitates and discriminate signals using counterstains like DAPI and PI.

Main Results:

• The protocol effectively disaggregates and concentrates bacteria from natural freshwater and marine samples.
• ELFA-labeled bacteria and precipitates are resistant to sample processing and can be stored for up to four months without significant loss.
• Flow cytometry successfully distinguishes ELFA-positive bacteria from non-labeled cells, with high recovery rates.

Conclusions:

• The developed flow cytometry protocol offers a robust and efficient method for assessing bacterial phosphatase activity at a single-cell level.
• This technique enhances the ability to study the physiological responses of heterotrophic bacteria to phosphorus limitation in aquatic ecosystems.
• The method's compatibility with various sample types and long-term storage capabilities makes it a valuable tool for microbial ecology research.