Isolation of Previously Uncultured Slow-Growing Bacteria by Using a Simple Modification in the Preparation of Agar Media

Most microorganisms living in the environment have yet to be cultured, owing at least in part to their slow and poor propagation properties and susceptibility to oxidative stress. Our previous studies demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving the phosphate and agar separately (termed "PS" medium), can greatly improve the culturability of microorganisms by mitigating oxidative stress compared with the use of "PT" medium (autoclaving the phosphate and agar together). Here, we attempted to isolate phylogenetically novel bacteria by combining PS medium with prolonged cultivation. After inoculation with forest soil or pond sediment samples, significantly more colonies appeared on PS medium than on PT medium. A total of 98 and 74 colonies that emerged after more than 7 days of cultivation were isolated as slow growers from PS and PT media, respectively. Sequencing analysis of their 16S rRNA genes revealed that the slow growers recovered from PS medium included more phylogenetically novel bacteria than those from PT medium, including a strain that could be classified into a novel order in the class Alphaproteobacteria. Further physiological analysis of representative strains showed that they were actually slow and poor growers and formed small but visible colonies only on PS medium. This study demonstrates that the culturability of previously uncultured bacteria can be improved by using an isolation strategy that combines a simple modification in medium preparation with an extended incubation time. IMPORTANCE Most microbial species inhabiting natural environments have not yet been isolated. One of the serious issues preventing their isolation is intrinsically slow and/or poor growth. Moreover, these slow and/or poor growers are likely to be highly sensitive to environmental stresses, especially oxidative stress. We reported previously that interaction between agar and phosphate during autoclave sterilization generates hydrogen peroxide, which adversely affects the culturability of environmental microorganisms, in particular, slow-growing organisms vulnerable to oxidative stress. In this study, we successfully isolated many slow-growing bacterial strains with phylogenetic novelty by simply modifying their cultivation on agar plates, i.e., autoclaving the phosphate and agar separately. The current limited repertoire of culture techniques still has room for improvement in the isolation of microorganisms previously considered unculturable.