Enumeration of marine microbial organisms by flow cytometry using near-UV excitation of Hoechst 34580-stained DNA

Abstract

A flow cytometry method for enumerating marine heterotrophic bacteria and phytoplankton in a living or preserved sample using a low power solid state near-ultraviolet laser is described. The method uses Hoechst 34580 to stain DNA in microbial cells in seawater samples. This stain is optimally excited at 375 nm unlike the similar Hoechst 33342, which requires ~ 350 nm excitation only available on more expensive lasers. Phytoplankton abundances from the Hoechst 34580 method are comparable to those of unstained samples and when analyzed by the Hoechst 33342 staining method. With this new method, nonpigmented marine bacteria and phytoplankton abundances are obtained simultaneously in a single sample as the Hoechst emission wavelength (~ 450 nm) is well separated from the emission wavelengths of chlorophyll and phycoerythrin fluorescence. Bacteria abundances are similar between this new method and those obtained with established Hoechst 33342 and SybrGreen I methods. Precision estimates (coefficient of variation) on populations with abundances near ~ 105 cells mL−1 are 1–3%, increasing to 3–9% at lower cell concentrations of 103 cells mL−1. The Hoechst 34580 method is simple, requiring no heating or pretreatment with RNAse, can be used on unpreserved and formaldehyde-preserved cells, and is amenable to at-sea use with portable, compact, low power-requiring flow cytometers.