Chapter 1 : New Approaches to Microbial Isolation

Abstract:

Metagenomics approaches promised to provide access to the uncultivated species while bypassing the problem of uncultivability. Significant departures from conventional techniques were clearly in order, and indeed, the new technologies substantially diverged from the cultivation tradition by adopting single-cell and high-throughput strategies, better mimicking the natural milieu and increasing the length of incubation and lowering the concentration of nutrients. This chapter describes the principle of these methods, details their application and the initial results of this application, and summarizes the lessons learned. Cultivation of novel species inside diffusion chambers is a welcome development, but their properties and abilities could be fully studied and utilized only if they could be adapted for growth in vitro. The authors therefore examined whether repetitive cultivation in a series of generations of diffusion chambers facilitated domestication of the grown strains, and discovered a positive correlation between the number of cultivation rounds in situ and the probability of obtaining a variant capable of growth in vitro. In a proof-of-concept study, the authors used the isolation chip (ichip) to grow marine water column and soil microorganisms, recorded the colony count, and compared the rRNA gene diversity of ichip-grown microorganisms and their colony count with microorganisms from parallel incubations in standard petri dishes. A reverse use of the diffusion chamber leads to a different type of cultivation (trap method), which selectively enriches for new and rare filamentous actinomycetes. This makes the trap method particularly useful for the discovery of novel secondary metabolites.