Enrichment of the subcultured microcolonies with candidate feeder organisms from the original mixed cultures was found to facilitate the growth of the microcolony-forming bacteria. Flow cytometry and cell sorting (FACS) is a method with numerous applications in microbiology (Alvarez-Barrientos et al., 2000). In an effort to cultivate as-yet-uncultivated taxa, Zengler et al. (2002) used gel microdroplets to encapsulate single bacterial cells (from dilutions of mixed environmental samples), which then formed microcolonies in situ. Based on characteristic light-scattering properties, any microdroplets
that contained microcolonies (as opposed to single or no cells) were detected this website and sorted by FACS, and subsequently analysed phylogenetically. When the intention is to detect and sort specific bacterial species, however, target-specific fluorochrome-labelled antibody or oligonucleotide probes are usually required. Whereas antibody-conjugated probes may preserve cellular viability, oligonucleotide probes do not, preventing the subsequent cultivation of sorted cells. Although FACS of ‘unculturable’ bacterial cells may not therefore directly lead to their cultivation, FACS in conjunction with whole-genome amplification has been used to obtain a partial genome sequence for a member of
the TM7 phylum (Podar et al., 2007). Knowledge of the genomes of as-yet-uncultivated organisms will help characterize these species and provide clues MG-132 datasheet that will aid their in vitro cultivation in the future. For example, genomic analysis of ‘Candidatus Pelagibacter ubique’ has revealed a deficiency of the genes 4��8C that are necessary for assimilatory sulphate reduction in the production of sulphur, which is essential for biosynthesis in aerobic marine bacteria (Tripp et al., 2008). The
micromanipulation of single bacterial cells for their isolation in pure culture has potential applications for the isolation of ‘unculturable’ bacteria (Frohlich & Konig, 2000). Optical tweezers, in the form of an infrared laser, are used to trap and isolate single cells within a cell separation unit from where they are ultimately transferred to growth media for cultivation. This method was used successfully by Huber et al. (1995) to isolate a previously uncultivated archaeal strain following visual recognition of its cellular morphology from targeted whole-cell hybridization. Raman tweezers, as used by Huang et al. (2009), involve a similar technique of optical trapping differing only in the method of cell recognition, which is based on the characteristic profile of spectral peak shifts within the Raman spectrum of individual cells. It is clear that there are many approaches to the cultivation of as-yet-uncultivated bacteria. Furthermore, the use of combinations of techniques has proven successful on several occasions. For example, Nichols et al.
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