Visualization and enumeration of marine planktonic archaea and bacteria byusing polyribonucleotide probes and fluorescent in situ hybridization

wFluorescent in situ hybridization (FISH) using rRNA-specific oligonucleoti de probes has emerged as a popular technique for identifying individual mic robial cells. In natural samples, however, the signal derived from fluor-la beled oligonucleotide probes often is undetectable above background fluores cence In many cells. To circumvent this difficulty, we applied fluorochrome -labeled polyribonucleotide probes to identify and enumerate marine plankto nic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identifica tion and enumeration of planktonic cells to ocean depths of 3,400 m, Quanti tative whole-cell hybridization experiments using these probes accounted fo r 90 to 100% of the total 4',6-diamidino-2-phenylindole (DAPI)-stained cell s in most samples. As predicted in a previous study (R. Massana, A. E. Murr ay, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50-56, 19 97), group I and II marine archaea predominate in different zones in the wa ter column, with maximal cell densities of 10(5)/ml. The high cell densitie s of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, renderi ng them easily detectable,vith polyribonucleotide probes. These results imp ly that the majority of planktonic cells visualized by DAPI do not represen t lysed cells or "ghosts," as was suggested in a previous report.