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

Abstract

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 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. 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.

Improved Isolation of Uncultured Anaerobic Bacteria using Medium Prepared with Separate Sterilization of Agar and Phosphate

“…Betaproteobacteria were more abundantly isolated from PS medium, whereas Actinobacteria were more abundant in PT isolates. These results are consistent with our previous findings, which were obtained using soil and freshwater sediments as the microbial sources (Tanaka et al, 2014;Kato et al, 2018). The majority of the isolates recovered under anaerobic conditions (nitrate reduction and fermentation conditions) were classified into Proteobacteria, Actinobacteria, Bacilli, and Clostridia.…”

“…The number of CFUs obtained under aerobic conditions was significantly higher for the PS protocol than for the PT protocol ( Fig. 1A), which is consistent with our previous findings (Tanaka et al, 2014;Kawasaki and Kamagata, 2017;Kato et al, 2018). Furthermore, CFU counts were 2to 3-fold higher on plates prepared using the PS protocol under nitrate reduction and fermentation conditions ( Fig.…”

“…We demonstrated that the separate sterilization of agar and phosphate (termed the "PS" protocol, where "S" indicates "separate") minimized ROS generation, which improved the culturability of environmental microorganisms. The PS protocol has been shown to improve the culturability of a wide range of microorganisms, including slow-growing heterotrophs (Kato et al, 2018), hard-to-culture Actinobacteria (Adam et al, 2018), and alkane-degrading bacteria (Zheng et al, 2018). Furthermore, many research groups reported the isolation of novel bacterial taxa using the PS protocol (Nishioka et al, 2016;Huang et al, 2017;Kitzinger et al, 2018;Rilling et al, 2018).…”

Cultivatingin situtriggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

“…Several studies have been conducted to determine the reasons for microbial uncultivability, such as inoculum size (55) and phosphate-catalyzed hydrogen peroxide formation from agar (12, 13, 56). A few attempts have been made to discover microbial interactions via signaling molecules that promote the growth of specific microbial types.…”

“…Standard agar plating as a conventional method of cultivating microorganisms is limited because a significantly low proportion (usually less than 1%) of the plated microbes readily form visible colonies on the agar plates, thus leading to plate count anomalies (6, 7). To overcome the limitations, much effort has been devoted to developing alternative approaches, including physically separating cells to decrease competition or inhibitors (8–10), using modification to prepare agar media, using alternative gelling agents or antioxidants to minimize unfavorable compounds (11–13), and adding signal molecules or cocultivating with recruiter organisms to better reflect the natural environment (14–16). Nevertheless, most postulated extant microbes in nature remain uncultivated; thus, other essential factors for microbial cultivation that exist in nature are likely absent from those artificial conditions.…”