Previous Article | Next Article ![]()
Applied and Environmental Microbiology, July 2000, p. 3037-3043, Vol. 66, No. 7
Skidaway Institute of Oceanography, Savannah,
Georgia 31411,1 and Georgia Institute of
Technology, Environmental Engineering, Atlanta, Georgia
303322
Received 28 January 2000/Accepted 28 March 2000
rRNA-targeted oligonucleotide probes have become powerful tools for
describing microbial communities, but their use in sediments remains
difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the
quantitative extraction of cells from formalin-preserved salt marsh
sediments. Resulting cell extracts are amenable to membrane blotting
and hybridization protocols. Using this procedure, the efficiency of
cell extraction was high (95.7% ± 3.7% [mean ± standard
deviation]) relative to direct DAPI (4',6'-diamidino-2-phenylindole)
epifluorescence cell counts for a variety of salt marsh sediments. To
test the hypothesis that cells were extracted without phylogenetic
bias, the relative abundance (depth distribution) of five major
divisions of the gram-negative mesophilic sulfate-reducing delta
proteobacteria were determined in sediments maintained in a tidal
mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide
probes were utilized. The apparent structure of sulfate-reducing
bacteria communities determined from whole-cell and RNA extracts were
consistent with each other (r2 = 0.60),
indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural
heterogeneity of sediment microbial communities and methodological
artifacts. The relative distribution of sulfate-reducing bacteria was
similar to that observed in natural marsh systems, providing
preliminary evidence that the mesocosm systems accurately simulate
native marsh systems.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Whole-Cell versus Total RNA Extraction for Analysis
of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide
Probes in Salt Marsh Sediments
*
Corresponding author. Mailing address: Skidaway
Institute of Oceanography, 10 Ocean Science Circle, Savannah, GA 31411. Phone: (912) 598-2308. Fax: (912) 598-2310. E-mail:
frischer{at}skio.peachnet.edu.
This article has been cited by other articles:
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»