Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

TLDR

Flow cytometry, especially bench‑top instruments coupled with fluorescent nucleic‑acid stains, is rapidly becoming a routine method for rapidly quantifying microbial abundance in lakes and oceans, offering cell‑specific measurements at scale that surpass epifluorescence microscopy. The study aims to illustrate how flow cytometry can differentiate photosynthetic from non‑photosynthetic prokaryotes, measure cell size and nucleic‑acid content, assess relative activity and physiological state, and inform the role of microbes in aquatic carbon cycling. The authors review the dyes, staining protocols, and bench‑top flow‑cytometer equipment used to count planktonic bacteria, addressing routine challenges such as fixation, staining, and absolute counting, and demonstrate how these methods enable differentiation of prokaryotic types, size and nucleic‑acid measurements, and activity assessments.

Abstract

Flow cytometry is rapidly becoming a routine methodology in aquatic microbial ecology. The combination of simple to use bench-top flow cytometers and highly fluorescent nucleic acid stains allows fast and easy determination of microbe abundance in the plankton of lakes and oceans. The different dyes and protocols used to stain and count planktonic bacteria as well as the equipment in use are reviewed, with special attention to some of the problems encountered in daily routine practice such as fixation, staining and absolute counting. One of the main advantages of flow cytometry over epifluorescence microscopy is the ability to obtain cell-specific measurements in large numbers of cells with limited effort. We discuss how this characteristic has been used for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content, and for estimating the relative activity and physiological state of each cell. We also describe how some of the flow cytometrically obtained data can be used to characterize the role of microbes on carbon cycling in the aquatic environment and we prospect the likely avenues of progress in the study of planktonic prokaryotes through the use of flow cytometry.