In Situ Isolation and Culturing of Recalcitrant Soil Bacteria using an Isolation Chip (iChip).

Ecosystems harbor millions of environmental microbes, some of them capable of producing biotechnologically relevant products such as enzymes, vitamins, amino acids, organic acids, anti-parasitic agents, and antimicrobial agents. Most of these environmental bacteria are deemed "unculturable" as the laboratory culturing methods fail to meet their nutritional/environmental requirement. Moreover, as they coexist in nature, they may also be dependent on a nutrient/metabolite produced by another member of the microbial community. Bringing the "uncultured" microbial diversity into the culture will present an opportunity to explore the vast array of bioactive products they may encode. This article describes the implementation of the iChip technology, a multichannel diffusion chamber developed for a high-throughput, in situ culturing of the "unculturable" bacteria. Given the global menace of antimicrobial resistance, the article provides a detailed protocol for in situ cultivation and recovery of isolates from the microbial 'dark matter' capable of producing antimicrobial agents, as an example. This protocol describes the steps involved, right from the soil sample collection step to the recovery of isolates, and highlights the plausible factors that may influence the success of isolation. The use of this technology not only facilitates the isolation of otherwise "unculturable" bacteria for antibiotic discovery but also enables researchers to delve into the complex soil ecology, which can then be tapped into for a myriad of other applications.