COURTESY OF PAMELA SCHNUPF. NATURE, 520:99-103, 2015.
In 2001, Nicole Dubilier, a marine biologist at the Max Planck Institute for Marine Microbiology in Bremen, Germany, made a surprising discovery—two symbiotic bacterial species living inside a gutless marine worm, Olavius algarvensis. To better understand the unique relationship among the three species, Dubilier set out to culture the two symbionts. But nearly 15 years later, she has yet to successfully grow the bacteria in the lab.
Her best attempt kept the microbes alive for about 10 months, Dubilier says, but then the culture “just died on us. . . . It’s a kamikaze project. How long can you have someone put in all their effort if it’s constantly unsuccessful?”
Dubilier is hardly alone in her plight. A heaping teaspoon of soil or a shot of ocean water may contain as many as one million bacterial species. Many of them are potential gold mines of chemicals and metabolites with medicinal, engineering, and energy applications. But when researchers have tried to culture these microbes in the lab, only a minority of cells form colonies. Clearly, nutrients, a carbon source, and time are usually not enough to coax bacteria isolated from the wild to grow in a laboratory setting. So what’s the missing ingredient?
If we can’t grow them, we don’t have the access to these organisms that rule our biosphere.—Kim Lewis,
“It’s a significant intellectual teaser,” says Slava Epstein, a microbial ecologist at Northeastern University in Boston, “why, after 150 years of the sweat and blood of smart, talented people, we can cultivate only a small proportion of microbes.”
In 1873, Joseph Lister first introduced serial limiting dilution of bacteria in liquid medium to achieve a pure culture. Then in the 1880s, Nobel laureate Robert Koch invented methods for growing pure bacterial cultures on solid media and his laboratory assistant, Julius Petri, created the round, flat, stackable plate that is still a fixture in microbiology labs. For almost as long, scientists have struggled to cultivate newly identified soil or marine microbes. “I wanted to trace the [history] of this phenomenon,” says Epstein. “But the search became difficult because soon I was tracking references from an era where references were done very differently, at a time when Pasteur was quoted as a contemporary. It’s that old.”
In 1985, microbiologists Allan Konopka of the Pacific Northwest National Laboratory and James Staley of the University of Washington dubbed this gap between the number of bacterial cells that could be directly counted in an environmental sample and those that could be reproducibly cultivated “The Great Plate Count Anomaly.” 1 More recently, researchers have applied metagenomics approaches, extracting and sequencing all microbial DNA from an ecological sample, and have revealed tens of thousands of new species, causing the gap to widen exponentially. One oft-cited statistic posits that only 1 percent of bacterial species in an environmental sample can be cultured, although microbiologists say this is more a symbol of the inability to culture most bacterial species rather than a number rooted in rigorous science. (See sidebar here.) “I really hate the statement that 1 percent are culturable. The [actual proportion] depends on the environment,” says Karsten Zengler, a microbiologist at the University of California, San Diego (UCSD). Either way, the true number of bacterial species is impossible to calculate without a better estimate of the total number of species around the globe.
Researchers are working hard to improve their chances of culturing fussy bacteria and hope to continue mining the world’s bacterial diversity for antibiotics and other therapeutic compounds, as well as for biotechnological advances. For example, the recently discovered CRISPR—an antiviral defense system found in many bacteria—has been widely adapted as a genetic-engineering tool. “If we can’t grow them, we don’t have the access to these organisms that rule our biosphere,” says Northeastern University microbiologist Kim Lewis.
“The need to cultivate organisms is greater now than ever,” agrees Tom Schmidt, who studies the ecology and evolution of microbes at the University of Michigan. “We need the whole organism to understand how it behaves in the environment and what organisms it interacts with; we can’t know that from just the parts list the genome provides.”
No guts, no glory
COURTESY OF PAMELA SCHNUPFOne reason that only a fraction of microbial species has been successfully grown in the lab is simply that most researchers aren’t willing to try. Microbiologists typically stick to easily grown bacteria such as E. coli, simply inserting genes from other microbes into this genetically malleable model organism. “Cultivation is a bit of a dying art,” says Zengler. “It can be a tedious, time-consuming, and often frustrating process.”
Dubilier agrees. “[Cultivation] is not what young scientists are interested in—it’s not sexy. They want to do the omics.”
But a few stubborn researchers are taking up the challenge. And for some types of microbes that don’t initially grow well using standard laboratory media, methodically offering the bacteria different culture conditions can pay off. David Fredricks, who studies how human microbiota affect health at the Fred Hutchinson Cancer Research Center in Seattle, was able to grow the majority of bacterial species found in human vaginal samples by plating them on a half dozen different media types kept in either aerobic or anaerobic conditions. Of course, patience, attention to detail, and a dissecting microscope were required, he notes, as some of the species formed only tiny colonies that were invisible to the naked eye.
Besides choosing the right growth medium, researchers also need to consider how bacteria are housed. What does not work well—at least for growing bacteria from vaginal fluid, says Fredricks—is growing colonies from single cells separated in wells, as is standard practice in clinical microbiology. That’s because it’s not just the nutrient and metabolite components of the medium that affect bacterial growth, but also signaling molecules and, potentially, physical signals that microbes get from their neighbors. McMaster University’s Michael Surette, whose lab has cultivated many of the bacteria found in both healthy and diseased human respiratory tracts, credits his group’s success to the use of unrestrictive culture conditions that allow interaction between species on standard media plates. “We treat each plate as a community rather than picking individual colonies.”
But too much interaction can be a bad thing, adds Surette. A little physical separation is important to give slow-growing species a chance to replicate in a dish filled with faster-growing microbes. “If you separate the bugs even by a few millimeters, the slow ones can still grow, yet the interactions within and between colonies are preserved,” Surette says. “It’s a trivial thing, but it’s powerful if you want to culture.”
Some bacteria are particularly finicky, however, and no combination of culture conditions and cell housing seems to work. In such cases, researchers must get creative. A few years ago, as a postdoc in microbiologist Philippe Sansonetti’s lab at the Pasteur Institute in Paris, Pamela Schnupf decided to culture segmented filamentous bacterium (SFB), a common gut bacterium in mammals. While SFB was well characterized in the 1970s, no successful attempts to culture the bacterium had been reported, and by the 1990s, the field had lost interest. Schnupf’s motivation to try again stemmed from the recent discovery that the bacterium is a key modulator of host immunity.2 “It acts as an educator of the immune system partly because it has this pathogenic pattern of attachment, and colonizes a niche different than other bacteria of the microbiota,” she says.
Schnupf and her colleagues initially poured over older SFB literature that cited failed cultivation attempts and undertook a brute-force approach, trying various conditions that might support SFB’s growth. But when nothing proved successful, Schnupf turned to the bacterium’s DNA for help.
Cultivation is a bit of a dying art. It can be a tedious, time-consuming, and often frustrating process.—Karsten Zengler, University of California, San Diego
“It was clear that SFB needed a rich environment because its genome was missing many biosynthesis pathway enzymes to make nucleotides and vitamins,” she recalls. “It also likely needed iron because it had genes for several iron transport systems.” Moreover, SFB’s genes suggested that, despite being an anaerobe, the bacterium could withstand low levels of oxygen, to which it is probably exposed in the small intestine. Schnupf took all of this information into account as she created a laboratory version of SFB’s in vivo niche: a human or murine cell line and tissue culture medium along with a rich array of nutrients regularly used for growing bacteria, spiked with extra iron, all cultured at a 1.5 percent oxygen concentration. Although it took several years to get the right cocktail, Schnupf was finally successful where others had failed for nearly half a century.3
“This was a pretty big breakthrough,” says Jonathan Dworkin, a microbiologist at Columbia University. “Before, if you wanted to work with SFB, you had to purify it from mouse poop. It was doable, but not very good for making rapid progress.”
COURTESY OF MICHAEL SURETTEAside from the singular difficulty of getting SFB to thrive in the lab, microbiologists have generally had reasonably quick success culturing human-derived microbes, at least in comparison with species isolated from environmental samples. More than half the members of the human microbiome can now be cultured, while less than 1 percent of bacterial species found in the wild have been grown in the lab, according to some estimates.
Culturing environmental microbes is difficult because many bacteria in the soil exist in a dormant state—a survival mechanism employed when nutrients are scarce and conditions adverse. Transferring these bacteria to a rich medium does not necessarily entice them to grow, and researchers know little about how to coax dormant bacteria out of their slumber. “If nutrients are the factors that wake up dormant cells, we would have discovered these by now, but that is not the case,” says Epstein.
Unlike hibernating bears or plants that respond in a predictable fashion to the changing environment, microbes may switch from dormancy to growth in a stochastic manner, he says. Epstein dubbed this idea “the scout model” in 2009.4 If a bacterium awakes and conditions are poor, it dies; the “scout” is sacrificed for the sake of the population. If conditions are favorable, the scout forms a new population and may even relay signals for its dormant neighbors to wake up. Epstein’s lab has provided evidence for this mechanism in laboratory isolates of E. coli and Mycobacterium smegmatis, as well as in a handful of species collected in the field.5,6 Dworkin has also shown that the spore-forming Bacillus subtilis goes through a similar random waking process.7
But even the stochastic model does not explain why the majority of bacteria cannot be cultivated in petri dishes, says Epstein. “The implication of this model is that populations that are massively growing at the time of sampling should be easier to culture than rarer ones, but in fact the reverse is true,” he says. “Typically, we can only cultivate a minor component of natural communities, and it’s exactly the abundant communities that are missing from laboratory plates.” A clade of marine bacteria called SAR11, for example, accounts for as much as one-quarter of the bacteria in seawater but has generally evaded cultivation.8
Epstein suspects that there may be a delay in bacterial growth as the microbes adjust to the alien conditions of the lab. The idea is that, while scout cells do randomly wake up to test the waters, they then adapt to their new environment by undergoing changes in gene expression, analogous to the process of differentiation in the cells of multicellular organisms. This delay in growth after a bacterial colony is transferred from one environment to another was noted by French biologist and Nobel laureate Jacques Monod in the early 1940s.9 “Monod described that E. coli exhibit a lag when transitioning from growth on glucose to lactose,” says Epstein. “But the distance between environmental and laboratory petri-dish conditions is huge, and adaptation may take a longer time, during which we pronounce the cells dead or uncultivable.”
© AL GRANBERGTo capture the efforts of Fredricks, Schnupf, Surette, and other microbiologists who’ve worked to culture fastidious bacterial species, Matthew Oberhardt, postdoctoral fellow in Eytan Ruppin’s laboratory at the University of Maryland Institute for Advanced Computer Studies, has been building a large database of detailed strain and media combinations for growing cultivable microorganisms. He hopes the data will serve as a source of insight into culturing principles, which might then be turned around and used for prediction. “I saw this as an opportunity to find principles underlying the media, to improve culturing conditions, and bring the art of culturing bacteria to high-throughput data,” he says.
Meanwhile, Epstein’s laboratory uses a multiwell diffusion chamber that it created in 2002, hoping to strike a balance between allowing slower-growing species to compete against more dominant strains and ensuring open communication among the bacterial community. The device, called the iChip, physically separates bacterial cells by a breathable membrane but surrounds them in their natural environment—the soil or marine water—allowing some contact and diffusion of factors and metabolites without the need to identify each species’ exact growth conditions.10 The diffusion also allows the multitudes of bacteria-produced factors to be preserved, as these compounds likely help culture species that prove difficult to grow under standard lab conditions.
© AL GRANBERGAnother growth tool, developed by UCSD’s Zengler around the same time Epstein’s group introduced the iChip, traps a single bacterial cell inside a tiny droplet made of a permeable gel, then uses flow cytometry to separate out those droplets in which a microcolony has formed.11 As with the iChip, the gel bead method isolates individual cells while preserving the interaction of the community, capturing both slow- and fast-growing bugs. The tool overcomes the sometimes painstaking microscopy needed to detect minute colonies that can get lost among the higher-density populations that grow like weeds on a petri dish. “Not every organism researchers are interested in grows to the density of E. coli,” says Zengler. “This allows the capture of these less-dense colonies.”
More recently, Zengler cofounded a company called GALT to develop and market high-throughput microbiology cultivation and screening tools. These tools address the bottleneck of cultivation, he says, by eliminating the labor-intensive and mostly slow process of isolating and growing bacteria in a controlled environment. And the demand for such products is growing, says Zengler, partly driven by the increased interest in the human microbiome as well as in soil microbes that may boost agriculture yields. “We need cost-effective and high-throughput cultivation methods to study microbiome interactions and develop microbes as therapeutics and to promote crop growth and yield.”
Access to microorganisms means access to their metabolites, says Gerry Wright, director of the Institute for Infectious Disease Research at McMaster University in Hamilton, Ontario. Wright has amassed a collection of 17,000 culturable species from previously under-sampled ocean and land environments and is now working to analyze the chemicals produced by these microorganisms. With a need for new antibiotics on the rise, researchers are hoping to discover antimicrobial molecules among the plethora of yet untapped bacterial biodiversity. In addition to antibiotics, there may be microbe-produced molecules that have immune modulatory, antiviral, or anticancer activities, he says.
The new tools allowing scientists to bring nature into the lab will perhaps convert some of the omics researchers into cultivation enthusiasts. The more bacterial species researchers are able to culture in the lab, the greater the chances of striking gold. “My view of the microbial world is as an iceberg,” says Dworkin. “We are seeing some idea of how these bacteria come up with solutions [for growth and communication], but we study few types of bacteria. We’re still missing a lot of interesting stuff.”
A NUMBERS GAME
There are about 12,400 cataloged bacteria species according to the List of Prokaryotic Names with Standing in Nomenclature, and most of these can be cultured to some extent. But these represent only a fraction of the presumed millions of species of microbes in the world, and only about half the known bacterial phyla have at least one cultured species.12,13
Although the exact numbers are unknown, it is often reported that only 1 percent of all known bacterial species have been successfully cultured in the lab. But no one has rigorously confirmed this estimate, says Slava Epstein, a microbial ecologist at Northeastern University in Boston. “It symbolizes a small number, no less but no more.” Indeed, many microbiologists agree that the 1 percent statistic is misleading. “Often, about 1 percent of bacterial cells from the soil will form colonies in a given experiment,” he says. “But that’s not to say 1 percent of species do.” It could be that most cells of a particular species will grow, while many other bacterial strains do not.
In marine environments, the percentage of bacterial cells that can be recovered and cultured is even smaller, about 0.01 to 0.1 percent. The easiest-to-grow species, it turns out, are those from the human gut. As many as one-half of the bacterial species identified in and on the human body will grow in the lab, says David Fredricks of the Fred Hutchinson Cancer Research Center in Seattle.
Other complications in calculating the proportion of bacterial species that can be cultured include defining a species at the genetic level and the fact that successful identification in metagenomic analyses depends on the sequence coverage. The greater the coverage, the greater the chance that more rarely represented sequences—from rarer bacterial species—will be detected, says Michael Surette of McMaster University and Farncombe Family Digestive Health Research Institute in Ontario, Canada. Conversely, low sequence coverage could underestimate the total number of species in a sample—and overestimate the percentage that has been successfully cultured.
Single-cell sequencing of a field sample may also fail to capture the fraction of cells that are in a state of dormancy, meaning even more species are missed. “There are lots of dormant cells recalcitrant to sequencing, and many surveys don’t use the proper procedures for getting DNA out of spores,” says Columbia University’s Jonathan Dworkin. “Just because sequencing technology keeps getting better does not negate the need for being able to isolate these dormant cells.”
- J.T. Staley, A. Konopka, “Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats,” Annu Rev Microbiol, 39:321-46, 1985.
- I.I. Ivanov et al., “Induction of intestinal Th17 cells by segmented filamentous bacteria,” Cell, 139:485-98, 2009.
- P. Schnupf et al., “Growth and host interaction of mouse segmented filamentous bacteria in vitro,” Nature, 520:99-103, 2015.
- S.S. Epstein, “Microbial awakenings,” Nature, 457:1083, 2009.
- S. Buerger et al., “Microbial scout hypothesis and microbial discovery,” Appl Environ Microbiol, 78:3229-33, 2012.
- S. Buerger et al., “Microbial scout hypothesis, stochastic exit from dormancy, and the nature of slow growers,” Appl Environ Microbiol, 78:3221-28, 2012.
- A. Sturm, J. Dworkin, “Phenotypic diversity as a mechanism to exit cellular dormancy,” Current Biology, 25:2272-77, 2015.
- M.S. Rappé et al., “ Cultivation of the ubiquitous SAR11 marine bacterioplankton clade,” Nature, 418:630-33, 2002.
- J. Monod, “Recherches sur la croissance des cultures bactériennes,” Paris: Hermann et Cie, 1942.
- T. Kaeberlein et al., “Isolating ‘uncultivable’ microorganisms in pure culture in a simulated natural environment,” Science, 296:1127-29, 2002.
- K. Zengler et al., “Cultivating the uncultured,” PNAS, 99:15681-86, 2002.
- N.R. Pace, “Mapping the tree of life: Progress and prospects,” Microbiol Mol Biol Rev, 73:565-76, 2009.
- M. Achtman, M. Wagner, “Microbial diversity and the genetic nature of microbial species,” Nat Rev Microbiol, 6:431-40, 2008.