The advent of ribosomal RNA sequence analysis 30 years ago revolutionized microbial ecology and significantly expanded our knowledge of microbial phylogenetic diversity (Woese and Fox, 1977). However, exclusive reliance on this single approach in environmental studies perpetuates certain assumptions that should be discussed and re-evaluated. These assumptions include (1) ribosomal RNA gene analyses can be used exclusively for the study of microbial diversity; (2) the number of colony-forming units (CFU) on nutrient media as a fraction of the total number of bacteria determined by microscopy is related to phylogenetic diversity and (3) only a fraction of environmental bacteria ‘species’ are culturable. Overlooking a century of cultivation history and encouraging use only of ribosomal approaches leads to significant gaps in microbial community diversity data. We demonstrate that cultivation methods are critical in microbial diversity studies and that they detect organisms undetected by molecular techniques. We thus caution against single-method approaches and posit that metagenomic techniques may be complemented by parallel culture libraries whenever diversity information is sought.

Use of the ribosomal approach alone in diversity studies originates in part with a review of its role in microbial ecology by Olsen et al. (1986) who stated, ‘…because molecules rather than organisms are isolated, the method is not limited to species that are amenable to laboratory cultivation’. Indeed, this technique has detected numerous microorganisms, for which no cultivated representatives are known (Stahl et al., 1984; Pace et al., 1986; Ward et al., 1990). Since these landmark studies, it has become a common practice to extract community DNA, amplify and clone fragments of ribosomal RNA genes and generate nucleotide sequences for comparison with those retrieved by other such studies. However, cultivation approaches are often set aside as researchers purport to describe microbial diversity by cultivating nothing more than a plasmid.

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