Cultivating the unculturable majority

Device to isolate pure bacterial cultures from the environment

It is widely recognized that the overwhelming majority of microorganisms cannot grow in artificial media that is prepared in laboratories. This issue was first realized over a century ago when the direct bacterial counts from environmental samples did not correlate with the number of resulting colony forming units (CFUs), a phenomenon known as “great plate count anomaly”. The problem is not only that very few colonies grow on the plates, but also that only a few species form colonies when compared to direct individual cell analysis of environmental samples. Cultivation-independent approaches, such as rRNA sequencing, have revealed that there remains an immense number of uncultivated species in nature. Thus, the vast complexity of the microbial world is still mostly unexplored and not understood.

We know that there are millions of microbial species that populate the world, but so far only a few have been characterized by more than just a sequence due to this inability to grow in the laboratory. A group of researchers at Northeastern University have now developed a device that allows researchers to cultivate a single species of bacteria that can then be studied and identified.

Over the last years there have been significant efforts made to overcome these limitations in cultivation. Previous research devices e.g. incorporated permeable membranes that allow sequestered bacteria to be exposed to the nutrients and molecules of their native environment. However, competition between species has so far limited the number of species of bacteria that could be isolated. 

The new device permits just a single bacterial cell to enter an inner chamber containing a food source, to which the only access is a microscopic passageway just slightly narrower than a single cell. The passageway is so small that the first cell to enter it gets stuck, blocking entry by any other cell or species. Once inside, this cell proliferates thereby filling up the inner chamber with a pure, single-species sample, since it is protected from any competition with other species.

In the paper, the team demonstrates the device's ability to separate mixtures of cell types in a laboratory setting by separating two different bacterial species whose cells have slightly different sizes (Escherichia coli and Pseudomonas aeruginosa). In a second experiment, they segregated  a combination of similarly sized but differently shaped species that commonly show up together in the marine environment (Roseobacter sp. and Pscyhoserpens sp). Finally, they used the device to separate cells of the same species that had been deferentially tagged to glow either red or green. In their final experiment they used the device to separate cells of the same species that had been differently tagged with either red or green fluorescent markers to validate the hypothesis that the cells grown inside the food chamber are actually daughters cells of the one cell caught in the entryway of the device. 

Who knows - a future form of this device might enable scientists to put more meaning to all those environmental DNA sequences and connect them to more information on individual organisms. A big step forward not only for the bacterial science community but also a shimmer of hope for a lot of fungi and protist researchers.