Although the microbiome teems with many, many individual species, few of them are easily grown in the laboratory. By refusing to thrive in conventional culture—a warm petri dish coated with nutrients—harmful microbes may stay “on the lam,” hiding among harmless or even beneficial microbes.
To isolate and cultivate individual bacterial species of interest, researchers at Caltech have devised alternative accommodations. These researchers, working in the laboratory of Rustem Ismagilov, have employed a specially designed glass chip with tiny compartments. The glass chip, called SlipChip, is a genetically targeted microfluidic device.
SlipChip is made up of two glass slides, each the size of a credit card, that have tiny etched grooves, which become channels when the grooved surfaces are stacked atop one another. When a sample—say, a jumbled-up assortment of bacteria species collected from a colonoscopy biopsy—is added to the interconnected channels of the SlipChip, a single “slip” of the top chip will turn the channels into individual wells, with each well ideally holding a single microbe. Once sequestered in an isolated well, each individual bacterium can divide and grow without having to compete for resources with other types of faster-growing microbes.
“It's difficult to recreate the complexity of the microbiome—the entire human microbial community—in one small plate,” explained Dr. Ismagilov. “The faster-growing bacteria take over the plate, and the slow-growing ones don't have a chance—leading to very little diversity in the grown sample.” Finding slow-growing microbes of interest is like finding a needle in a haystack, he added, but his group wanted to work out a way to “just grow the needle without growing the hay.”
In the Ismagilov lab, Liang Ma, a postdoctoral student, developed a method that combined microfluidics and on- and off-chip assays. The details appeared June 25 in the Proceedings of the National Academy of Sciences, in an article entitled “Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project’s Most Wanted taxa.”
The method, wrote the authors, “involves (i) identification of cultivation conditions for microbes using growth substrates available only in small quantities as well as the correction of sampling bias using a “chip wash” technique; and (ii) performing on-chip genetic assays while also preserving live bacterial cells for subsequent scale-up cultivation of desired microbes, by applying recently developed technology to create arrays of individually addressable replica microbial cultures.”
The second step described above gets around a Catch-22—the need to kill an organism in order to identify it via DNA sequencing, when the ultimate goal is to secure a living organism. “Liang solves this in a really clever way,” remarked Dr. Ismagilov. “He grows a compartment full of his target microbe in the SlipChip, then he splits the compartment in half. One half contains the live organism and the other half is sacrificed for its DNA to confirm that the sequence is that of the target microbe.”
To validate the new methodology, the researchers isolated one specific bacterium from the Human Microbiome Project’s “Most Wanted” list. The investigators used the SlipChip to grow this bacterium in a tiny volume of the washing fluid that was used to collect the gut bacteria sample from a volunteer. Since bacteria often depend on nutrients and signals from the extracellular environment to support growth, the substances from this fluid were used to recreate this environment within the tiny SlipChip compartment—a key to successfully growing the difficult organism in the lab.
After growing a pure culture of the previously unidentified bacterium, Ismagilov and his colleagues obtained enough genetic material to sequence a high-quality draft genome of the organism. Although a genomic sequence of the new organism is a useful tool, further studies are needed to learn how this species of microbe is involved in human health, Dr. Ismagilov noted.
“To our knowledge, this is the first example of targeted isolation of a high-priority member from the [Most Wanted] list, and is the first successful targeted cultivation from a complex biological sample of a previously uncultured taxon defined only by short reads from high-throughput sequencing of the 16S rRNA gene,” the authors concluded. “We envision that the microfluidics-based workflow described in this paper will be useful for conclusively testing hypotheses generated from culture-independent studies by providing pure cultures of biomedically and environmentally significant microorganisms.”