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Applied and Environmental Microbiology, December 1999, p. 5554-5563, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Visualization and Enumeration of Marine Planktonic
Archaea and Bacteria by Using Polyribonucleotide Probes and
Fluorescent In Situ Hybridization
Edward F.
DeLong,1,*
Lance
Trent
Taylor,1
Terence L.
Marsh,2 and
Christina
M.
Preston1
Monterey Bay Aquarium Research Institute,
Moss Landing, California,1 and
Center for Microbial Ecology, East Lansing,
Michigan2
Received 29 June 1999/Accepted 5 October 1999
Fluorescent in situ hybridization (FISH) using rRNA-specific
oligonucleotide probes has emerged as a popular technique
for identifying individual microbial cells. In natural samples,
however, the signal derived from fluor-labeled oligonucleotide probes
often is undetectable above background fluorescence in many cells.
To circumvent this difficulty, we applied fluorochrome-labeled
polyribonucleotide probes to identify and enumerate marine planktonic
archaea and bacteria. The approach greatly enhanced the sensitivity and
applicability of FISH with seawater samples, allowing confident
identification and enumeration of planktonic cells to ocean depths of
3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total
4',6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As
predicted in a previous study (R. Massana, A. E. Murray,
C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol.
63:50-56, 1997), group I and II marine archaea predominate in
different zones in the water column, with maximal cell densities of
105/ml. The high cell densities 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, rendering them
easily detectable with polyribonucleotide probes. These results imply
that the majority of planktonic cells visualized by DAPI do not
represent lysed cells or "ghosts," as was suggested in a previous report.
*
Corresponding author. Mailing address: Monterey Bay
Aquarium Research Institute, P.O. Box 628, Moss Landing, CA 95039. Phone: (831) 775-1843. Fax: (831) 775-1645. E-mail:
delong{at}mbari.org.
Applied and Environmental Microbiology, December 1999, p. 5554-5563, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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