Flow cytometric determination of bacterial abundance in lake plankton with the green nucleic acid stain SYTO 13

We have developed a protocol for the cytometric enumeration of bacterioplankton in lakes with the nucleic acid stain SYTO 13; our protocol can be used with any standard bench-top flow cytometer equipped with an argon laser with excitation at 488 nm. The protocol performs well in the range of bacterial densities from 10(5) to 10(6) ml(-1). Estimates of bacterial abundance using the cytometric technique ranged from 72 to 141% of the epifluorescence estimates with DAPI staining. The average C.V. of replicate cytometric counts was 2.4%, and the average C.V. for cytometric counts in replicate water samples was 6.7%. The precision of cytometric counts was 10-fold higher than corresponding epifluorescence bacterial counts, and the sample processing time is decreased by 10-15-fold relative to the epifluorescence technique. This cytometric protocol can be applied to samples from a wide variety of lakes and offers an accurate, precise, and fast alternative to standard epifluorescence bacterial counts.