During the past over two decades, the use of culture-independent nucleic acid techniques, represented by ribosomal RNA gene cloning library analysis, has unveiled the tremendous microbial diversity that exists in natural environments
. In sharp contrast to this great achievement is the current inability to cultivate the majority of bacterial species or phylotypes revealed by molecular approaches. One of the major difficulties for microbial ecology is that conventional cultivation methods provide access to only a very small fraction of the microbial diversity and more than 99% of naturally occurring microbes are considered ‘unculturable’ on standard culture media
. Although recent new technologies such as metagenomics and metatranscriptomics can provide more functionality information about microbial communities
[2, 3], it is still important to develop the capacity to isolate and cultivate individual microbial species or strains in order to gain better understanding of microbial physiology and to apply isolates for various biotechnological applications
A new view is emerging among microbial ecologists that the majority of so-called ‘unculturable’ microbial species simply have not been cultured yet. In line with this view, more research is necessary to enhance the ability to culture microbes, in order to reduce the dependence on indirect and cumbersome metagenomic approaches
[5, 6]. Recent developments in improving traditional cultivation techniques have shown that some conventionally unculturable species can in fact be grown as pure cultures. Tamaki et al. reported that the use of gellan gum instead of agar as the solidifying agent could greatly improve the cultivability of novel microbes on solid media
[7, 8]. Kaeberlein et al. designed a diffusion chamber to grow previously uncultivated pure isolates of marine origin
, and the same strategy was also successfully used in cultivation of groundwater microorganisms
. Stevenson et al. achieved similar results with soil microbes by fine-tuning the oxygen concentration and nutrient levels
. Knowledge obtained through metatranscriptome analysis has also been used in directed cultivation of bacteria
. These examples shown that many microbial species can be cultured as long as the environments are optimized for growth.
Recently, some non-traditional cell-isolation technologies have been introduced to isolate targeted cells for pure culture cultivation. For example, Huber et al. used optical tweezers to track and isolate an extremophilic archaeon from a microbial community in a terrestrial hydrothermal vent field
. This method of single-cell manipulation provides a new way to grow pure microbial cultures from single mother cells, but it has the disadvantage that the identification and manipulation of the bacteria is an extremely labor-intensive process. By contrast, a high-throughput isolation method has been presented using encapsulated single bacteria in droplets of gel
, resulting in the successfully cultivation of pure cultures from marine microorganisms. Oligonucleotide probes were used to identify the species after isolation.
One of the remaining hurdles for bacterial cultivation is that fine-tuning the growth condition for any specific species is a daunting effort, especially with the widely diverse microbiota in the ocean and soil environments. Consequently, a high-throughput platform is urgently needed to perform trials of different cultivation conditions in parallel. Previously, several high-throughput microtiter-plate-based cultivation platforms have been developed for marine and aquatic water column bacteria, and these have contributed greatly to the successful cultivation of previously uncultivated bacteria
[15, 16]. Most recently, a chip-based version of a Petri dish and diffusion chamber has been developed to address the same purpose
[17, 18]. Microfluidic ‘lab-on-a-chip’ (LOC) devices have been used for co-cultivation of various bacterial strains and species
[19, 20]. These devices are complicated in structure, utility, and fabrication, and are therefore less useful for general microbiologists.
In this study, we developed a parallel cultivation set-up that incorporates streamlined processes and is compatible with downstream genomic analysis. It spontaneously isolates environmental bacteria into miniature incubation chambers from a mixed microbial community. The key component of the system is the microbe observation and cultivation array (MOCA), which uses a Petri dish that contains an array of droplets with an oil covering as cultivation chambers. During cultivation, the growth of bacteria across the droplet array can be monitored using an automated microscope, which can produce a real-time growth record. Compared with conventional cultivation methods, MOCA provides streamlined preparation, parallel cultivation, and real-time observation, and unlike other chip-based platforms
[16–19], MOCA does not require complicated engineering techniques or equipment for fabrication. We have found that bacterial growth across the droplet array has a high level of uniformity when the initial cell density is more than 10 cells/μl, and thus MOCA provides a novel platform for bioassay screening. When the cell occupancy in droplets is at the single-cell level, real-time image recording can be used to monitor the growth and morphological development of microcolonies derived from single bacterial cells. The droplet culture developed from a single bacterial cell can be transferred using a micropipette
 for bulk cultivation or further molecular analysis.