New Strategies for Cultivation and Detection of Previously Uncultured Microbes

Abstract

An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorumsignaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O 2 [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO 2 (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO 2 . A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.

Substrate-induced growth and isolation of Acidobacteria from acidic Sphagnum peat

“…The inoculated plates were incubated in the dark at 10, 15 or 20 1C for 1-2 months. The surveillance of isolation plates for the presence of target bacteria was done by means of plate-wash FISH, a procedure similar to plate-wash PCR described by Stevenson et al (2004) with the only difference that, instead of PCR with Acidobacteria-specific primers, we used FISH with Acidobacteria-specific probe. When development of the target bacteria had been detected Ombrotrophic bogs are wetlands with no contact to groundwater, so that the only source of mineral nutrients is atmospheric precipitation.…”

Phylogenetic diversity of Acidobacteria in a former agricultural soil

“…Although, Acidobacteria have generally been refractory to classical cultivation, recent developments in improving cultivation methods have yielded a number of new soil isolates affiliated with subdivisions 1-4 Sait et al, 2002Sait et al, , 2006Joseph et al, 2003;Stevenson et al, 2004;Eichorst et al, 2007). However, cultivationindependent approaches, based upon 16S rRNA gene sequence analysis, have revealed a greater diversity of Acidobacteria in soil, with subdivision 6 typically being most highly represented in clone libraries (Janssen, 2006).…”

Enhanced and continued degradation of microcystins using microorganisms obtained through natural media

“…The fact that bacteria isolated through artificial media often show limit removal efficiency or encounter failure in natural environment, urges researchers to optimize the isolation strategy, such as the use of agar media with little or no added nutrients and relatively long periods of incubation (Stevenson et al, 2004). Janssen et al (2002) use diluted nutrient broth as growth medium.…”

“…To bypass the bottleneck of microorganism isolation, measures are taken to modify the isolation media or procedure, including the use of agar media with little or no added nutrients and relatively long periods of incubation (Stevenson et al, 2004) and the diluted nutrient broth as growth medium (Janssen et al, 2002). These methods allow cultivation of previously uncultivated microorganism and have been successfully used in biodegradation test under laboratory conditions.…”