The Culture of ‘Unculturable’ Microbes

In my last post, I mentioned that there are a plethora of unstudied and undiscovered microbes hiding in plain sight, but do these unstudied microbes have to be rare or hiding in low numbers to remain unstudied? Sometimes unstudied microbes remain that way because we haven’t yet discovered how to bring them into the laboratory. Microbes that have no known method for culturing in the lab, called ‘unculturables’, represent a huge challenge in the field of microbiology.

Unculturable microbes are the majority.

The hard truth is that the microbes we can grow in the laboratory only represent a small fraction of the total microbial diversity that exist in nature. Many of these microbes play critical roles in the carbon and nitrogen cycles that allow our planet to sustain life. Additionally, these microbes likely produce novel chemicals and products that could have medical or environmental impacts.

How do we know these unculturable microbes have these abilities? Next generation sequencing technologies have enabled us to get a snap-shot of the blueprints or DNA of these ‘unculturable’ microbes straight out of the environment.

What is an unculturable microbe?

Even as early as 1911, microbiologists observed many more microbes through the microscope than they could grow on agar plates and referred to the problem as the “The Great Plate Count Anomaly” (Amann, J. 1911). Since the DNA of unculturable microbes is routinely found in samples, they must be growing in the environment somehow. In conclusion, we lack a proper understanding of which components of an environment are vital for growth in the lab.

Why do we have to grow them in the lab?

While advances in sequencing technology have taught us what exists, the DNA sequences alone tell us nothing of how these genes and pathways actually work. Much of our current understanding relies on extrapolations from well-studied microbes – commonly referred to as model organisms – we routinely grow in the lab. Relying on these microbes to understand new genes or pathways found in unculturables that are very dissimilar becomes very unreliable. Even research centered on the physiology of bacteria we know well and can grow in the lab – like Escherichia coli or E. coli – routinely generate new findings and remain the subject of much research to this day.

Culturing the unculterable: where do we start?

Natural environments can be complex and dissecting each chemically and biologically can be an extremely difficult task, however, microbiologists around the world are coming up with clever and purposeful experimental designs to circumvent the unculturable challenge.  

Design I: Bringing the environment into the lab.

As seen here, natural environments can be extremely complex. Instead of figuring out precisely what is important, scientists are bringing all the possible components into the lab! Photo Credit: Nuno-Gomes; cc licence.

As seen here, natural environments can be extremely complex. Instead of figuring out precisely what is important, scientists are bringing all the possible components into the lab! Photo Credit: Nuno-Gomescc licence.

The first approach is what Carl Zimmer cleverly referred to as ‘upgrading … Petri dishes’. For example, Kim Lewis’ group at Northeastern is having success in re-building the entire environment in aquaria, complete with natural seawater, sand, flora, and fauna. These scaled down environments don’t require scientists to have an intimate knowledge of the exact features needed to grow certain microbes while still allowing them access to generally difficult to culture bacteria species.

Sometimes you just need the water; using natural salt water itself as a growth medium has also allowed a widespread group of unculturable free-living bacteria such as the SAR11 clade of Alphaproteobacteria to be grown in the lab. Relying on the fact that this unculturable bacterium was one of the most abundant organisms present, Stephen Giovannoni's group at Oregon State University cleverly diluted their ‘grown’ water samples enough to eliminate the bacteria they didn’t want.

Design II: Sometimes, unculturables need a little help from their friends

What makes for good laboratory growth conditions? The above examples demonstrate that emphasis on isolation is not always ideal.  Some unculturables depend on the activity of other microbes and the secreted byproducts and molecules that result. This type of dependency means these organisms must be grown in the presence of others in a method called coculture.

An artists interpretation of the coculture method. Displayed is Cluster A Synergistetes dependency on other natural mouth microbes to grow on Petri dishes in the laboratory setting. Photo credit: Gabrielle Dowell.

An artists interpretation of the coculture method. Displayed is Cluster A Synergistetes dependency on other natural mouth microbes to grow on Petri dishes in the laboratory setting. Photo credit: Gabrielle Dowell.

For example, some variants of a common, photosynthetic microbe called Prochlorococcus requires the presence of other non-photosynthetic bacteria to grow on Petri dishes. Coculture requirements may also be more common than we know. Some microbes identified in the sequencing effort to identify human-associated microbes – called the human microbiome project – have been shown to require coculture. For example, many microbes that form the film that builds up on your teeth when you don’t brush them for a while, called dental plague, have been identified, but not cultured. Recently, however, unculturable isolates have been successfully grown in the lab using cocultures with other mouth microbes. Good news for dental hygiene enthusiasts.

In summary, the field of unculturable microbes represents a huge challenge for advancing our understanding of the microbial jungle. Lucky for us, clever microbiologists are making strides to overcome existing limitations to access this black box.

Liked this post? Then you should check out other posts about our mysterious microbial world and remember, stay hungry!