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Applied and Environmental Microbiology, July 2000, p. 3037-3043, Vol. 66, No. 7
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

Marc E. Frischer,1,* Jean M. Danforth,1 Michele A. Newton Healy,1,2 and F. Michael Saunders2

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.


* 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.


Applied and Environmental Microbiology, July 2000, p. 3037-3043, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.



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