Previous quantifications of microbial cells in deep subsurface sediments used generic DNA stain acridine orange and did not allow a separate quantification of bacterial and archaeal cells. In Leg 201 sediments, quantifications of bacteria and archaea were performed by quantitative PCR and by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Quantitative PCR of bacterial and archaeal 16S rRNA genes at Sites 1227 and 1230 indicated that bacterial 16S rRNA genes outnumber archaeal 16S rRNA genes by one to three orders of magnitude, with considerable site-to-site variability (Schippers et al., 2005). Quantifications with quantitative PCR by another team showed that the archaeal 16S rRNA genes constituted maximally 5%–10% of the total prokaryotic 16S rRNA gene population in the upper 10–20 m of the sediment column (Sites 1227 and 1230); the archaeal proportion dropped below detection limit at greater depths (Inagaki et al., 2006).
Direct counts of bacterial and archaeal cells with the highly sensitive CARD-FISH approach targeting rRNA within metabolically active cells (Pernthaler et al., 2002; Teira et al., 2004) indicated that bacterial cells outnumber archaeal cells in deep marine sediments (Schippers et al., 2005). Bacterial cells constituted ~10%–30% of the total AODC of prokaryotic cells; archaeal cells remained below the statistical detection limit of CARD-FISH counts (Schippers et al., 2005). However, other groups that quantified bacterial and archaeal populations in Leg 201 sediments using CARD-FISH found substantially higher archaeal numbers (Mauclaire et al., 2005; Biddle et al., 2006). Systematic methodological comparisons and experimental standardizations seem to be necessary to resolve these discrepancies.
The observation that CARD-FISH counts account only for 10%–30% of AODC indicates that only a minority of cells harbor detectable amounts of 16S rRNA, the molecule that is the target for CARD-FISH detection. Cellular ribosomal RNA content is roughly proportional to metabolic activity and nutritional state of microbial cells (Poulsen et al., 1993; Fukui et al., 1996; Molin and Givskov, 1999). In other words, the CARD-FISH counts quantify metabolically active, living cells, whereas AODC quantifies living, inactive, and even dead cells, as long as their cellular morphology and integrity remains intact. Thus, CARD-FISH counts give solid evidence for presently active subsurface bacterial and archaeal communities in the deep subsurface, in contrast to the "palaeome" concept that the subsurface accumulates inactive or fossil cells (or their DNA) as a record of past microbial activity (Inagaki et al., 2005).