Estimation of bacterial cell numbers in humic acid-rich salt marsh sediments with probes directed to 16S ribosomal DNA

The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating; cell numbers was examined and applie d to study the structure of a bacterial community in humic acid-rich salt m arsh sediments. Hybridizations were performed with membrane-bound nucleic a cids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate- reducing bacteria (SRB), DNA was extracted from sediment samples, and conta minating materials were removed by a series of steps. Efficiency of DNA ext raction was 48% based on the recovery of tritiated plasmid DNA added to sam ples prior to extraction. Reproducibility of the extraction procedure was d emonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extr acted DNA obtained with each probe to that obtained with a standard curve o f genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 x 10(7) to 2.5 x 10(9) (average, 1.1 x 10(9) +/- 5.2 x 10(8)) cells g of sediment( -1). In September, numbers of SRB detected ranged from 5.4 x 10(8) to 7.3 x 10(9) (average, 2.5 x 10(9) +/- 1.5 x 10(9)) cells g of sediment(-1). The capability of using rDNA probes to estimate cell numbers by hybridization t o DNA extracted from complex matrices permits initiation of detailed studie s on community composition and changes in communities based on cell numbers in formerly intractable environments.