Indeed, why, after 150+ years of microbiology as science, and gigantic efforts of countless microbiologists, have less than 1% of global microbial diversity has been cultivated? Have our previous efforts been lacking in discipline or is there something critical missing from the traditional approach altogether? It seems that, if we continued the tradition of conventional cultivation, which basically amounts to tinkering with the composition of nutrient media and incubation conditions, the progress would just be incremental. A radical change appears to require a fundamental departure from convention.
Diffusion chamber platform
Some years ago, together with Kim Lewis, presently at Northeastern University in Boston, we asked this question: is it possible to grow something, e.g. a microbial species, that has unknown growth requirements, without first resolving these unknowns? The answer is likely “yes,” because, assuming all species grow in nature, their cultivation might be possible via a reasonable imitation of the natural conditions conducive of this species’ multiplication. Note that in principle, the natural milieu can serve as an ultimate growth medium even if we do not know all its components. Considering microbial cultivation from this point of view suggests that, curiously, cultivation per se does not seem to be a problem: if a cell from a growing population is removed from its natural habitat, and then – instead of going to Petri dish – is returned back to the environment, chances are it will grow again. This growth could be equated with cultivation, except of course the results of such “cultivation” are inaccessible, for the newly “cultivated” species is (again) mixed with all its neighbors. Nonetheless, this suggests that the real problem is not necessarily how to grow environmental microorganisms – once more, this is already happening in their own environment – but rather how to contain this growth.
Following this logic, we placed environmental cells into a diffusion chamber with 0.03-µm pore-size membranes (Fig. 1A), and returned the chamber into the natural environment from where the cells were collected, so that diffusion would establish chemical communication between the cells and nature. A proof-of-concept growth experiment showed a 30,000% higher recovery rate of marine microorganisms over parallel cultures that utilized traditional techniques (standard Petri dish) techniques (Kaeberlein et al 2002; Fig. 2). Later we showed that that the species composition of the grown material is also markedly different between the chamber and Petri dish (Bollmann et al 2007).
We further reasoned that the same device could be used in a very different fashion, and enrich for different microorganisms. The idea is that, if a diffusion chamber is NOT inoculated with microorganisms, and contains sterile agar, and if the pores of the membranes are > 0.2-0.4 µm, then during this device’s environmental incubation filamentous, chain-forming, and mobile species should colonize the inner space (Fig. 2B). We confirmed this in a proof-of-concept study, and isolated a number of novel Actinobacteria, fungi, and other microorganisms from soils (Gavrish, et al. 2008). Note that the principle step, in situ enrichment, is a cornerstone of both the trap and diffusion chamber approaches, but the resulting culture collections are significantly different, making these methods complementary.
The use of the diffusion chamber has one main limitation: low throughput. To resolve this bottleneck, we designed a variant of the diffusion chamber, “the isolation chip”, or ichip for short, that consists of numerous micro-diffusion chambers containing single cells (Nichols et al. 2010). This modification enables the diffusion chamber-based approach to grow AND isolate environmental microorganisms into pure culture in a single step. The ichip is an assembly of flat plates containing multiple registered through-holes (Fig. 3). When the central plate is dipped into a cell suspension in a gelling agent (left panel), each through-hole captures a volume of suspension containing a certain number of cells. If the suspension is appropriately diluted, this number may be on average one cell per through-hole (central panel). Upon solidification of the agent, the cells are immobilized inside small gel plugs, and become “trapped” and isolated from each other. Sandwiching the plate between 0.03-µm pore size polycarbonate filters effectively transforms the loaded plate into an array of many diffusion mini-chambers (right panel).
Subsequent in situ incubation in the cell’s original environmental habitat provides the immobilized cells with their naturally occurring growth components. Since most mini-chambers contain single cells, they grow as pure cultures. In this way, cultivation/isolation of uncultivated species is achieved in large numbers, and in a single step.
One notable modification of the ichip in Fig. 3 is its highly miniaturized variant suitable for incubation in the human body, such as for example in the oral cavity (Sizova et al. 2012; Fig. 4).
Methods in development
Presently we are working on the next generation cultivation devices that will be able to autonomously sample individual microbial cells, grow them in their natural environment, and remotely transmit information about their properties – all with no participation of a microbiologist. We term this device Gulliver, because it will be able to travel to where microbiologists cannot – from the bottom of the ocean to the inside of human gut to extraterrestrial bodies.
T.Kaeberlein, Lewis, K., and Epstein, S.S. (2002). Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296: 1127-1129
Gavrish, E., Bollmann, A., Epstein, S. S., and Lewis, K. (2008) A trap for in situ cultivation of filamentous actinobacteria. J. Microbiol. Methods 72: 257–262.
Nichols,, D., Cahoon,N., Trakhtenberg, E.M., Pham, L, Mehta, A., Belanger, A., Kanigan, T., Lewis, K., and Epstein, S.S. (2010). Ichip for high-throughput in situ cultivation of “uncultivable” microbial species. Appl. Environ. Microbiol., 76: 2445-2450
MV Sizova, T Hohmann, A Hazen, BJ Paster, SR Halem, CM Murphy, NS Panikov, SS Epstein (2012) New approaches for isolation of previously uncultivated oral bacteria Applied and environmental microbiology 78: 194-203
The methods are summarized in several recent reviews:
Epstein, S.S., Lewis, K., Nichols, D., and Gavrish, E. (2010). New approaches to microbial isolation. In: Manual of Industrial Microbiology and Biotechnology, (eds.) R.H. Baltz, A.L. Demain, and J.E. Davies. Washington, DC, ASM, pp. 3-12.
SS Epstein, M Sizova, A Hazen (2013) New approaches to cultivation of human microbiota In: The Human Microbiota: How Microbial Communities Affect Health and Disease, John Wiley & Sons, Inc. pp 303-314