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

Abstract

SUMMARY: 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.

Transformation of iodate to iodide in marine phytoplankton driven by cell senescence

“…Bacterial abundances were determined by flow cytometry according to Gasol & Del Giorgio (2000). Samples were fixed with 0.2 µm prefiltered formaldehyde (2% final concentration) in 5 ml cryovials, deepfrozen in liquid nitrogen after a 30 min dark incubation, and stored at -80°C.…”

Morphology and DNA content of bacterioplankton in the northern Gulf of Mexico: analysis by epifluorescence microscopy and flow cytometry

“…Vertical and regional patterns in bacterioplankton diversity were addressed by quantification of morphologically distinguished bacteria using epifluorescence microscopy and of subpopulations with different apparent DNA content using flow cytometry. Previous studies on the apparent DNA content of heterotrophic bacteria led to the hypothesis that low-DNA bacteria are dormant or inactive and only high-DNA bacteria are active (reviewed in Gasol & del Giorgio 2000). The present study aimed to reveal natural distribution patterns of low-versus high-DNA bacteria in the oligotrophic open Gulf of Mexico and the eutrophic Mississippi River plume.…”

“…from heterotrophic bacteria, which can cause a significant overestimation of bacterial abundance (Campbell et al 1994, Sieracki et al 1995. The application of flow cytometry to aquatic microbial ecology and the recent development of blue-light excited cyanine dyes (Lebaron et al 1998) helped to establish new protocols for cytometric quantification of heterotrophic bacteria, which turned out to be much faster and more precise than traditional microscopic methods (see review in Gasol & del Giorgio 2000). Phototrophic prokaryotes, Prochlorococcus spp.…”

“…Phototrophic prokaryotes, Prochlorococcus spp. and Synechococcus spp., can be distinguished from heterotrophic bacteria by their chlorophyll autofluorescence in cytometric analyses (Li et al 1995, Marie et al 1997, Gasol & del Giorgio 2000.…”

Changes in marine bacterioplankton phylogenetic composition during incubations designed to measure biogeochemically significant parameters

“…Prokaryotic counts were performed with a FACSCalibur flow cytometer after staining with Syto13 (Gasol and del Giorgio 2000). Cytometry data were also used to calculate the average cell volume and the percentage of high DNA bacteria (%HDNA).…”