Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Bacteria need not only to sense the presence of things like nutrients and toxins, but also to know in which direction they lay—the better to gravitate toward or away from them. By studying the response of these microbes to chemical gradients, researchers can gather a wealth of information about proclivities and sensitivities to, and antipathies for, the substances both individually and in combination with other stimuli—information that can help unravel puzzles as diverse as pathogenesis, bioremediation and biofilm formation.
Numerous assays have been developed over the years (typically based on agar plates) to investigate how motile bacteria respond to chemical gradients. Yet in most of these, it has been difficult to accurately manipulate and quantitate the gradient. Furthermore, they often required a choice between obtaining statistically meaningful results from a population and observing a few individual cells.
Microfluidics circumvents many of those difficulties, enabling researchers to simplify manipulation and control of the environment on the bacterial scale.
Ready for prime time?
The vast majority of research papers on the use of microfluidic technologies have been published in engineering journals. Most microfluidics papers published in biology and medical journals are the result of collaborations with engineers, writes David Beebe and colleagues in a recent review [1]. Part of the reason, they write, is that a “killer application”—research essential to the field that can only be done with microfluidics—is yet to emerge.
Yet despite this, Roman Stocker, associate professor of civil and environmental engineering at Massachusetts Institute of Technology (MIT), points out two ways in which “you can do a whole lot more with microfluidics.”
First, after exposing bacteria to a precisely defined gradient, “we can scan a channel the size of a glass slide and get the positions of thousands, sometimes tens of thousands of bacteria. So [as] you can imagine, your statistics are extremely robust,” he says. Traditional swarming or soft agar assays, on the other hand, only give a sense of the ring of expansion of chemotaxis and often provide only a yes/no answer to the question of whether bacteria are attracted to a given substance, relative to control.
Even compared to a relatively quantitative capillary assay—in which a chemoeffector-loaded capillary tube is inserted into a bacterial plate, allowing the bacteria to swim up the newly formed gradient and into the tube—microfluidics is several orders of magnitude more sensitive. That makes a difference, Stocker says, “because natural environments often have exceedingly low concentrations of chemicals”—often far lower than may be assayed by typical motility assays.
Second, “you can image behavior, so you don’t just see a population-scale response, you can really see what individuals are doing,” Stocker points out. “You can track cells frame to frame and ask, ‘Do they speed up?’ ‘Do they slow down?’ You can see whether they achieve chemotaxis by turning in a certain way as opposed to another.” Recently, for instance, Stocker’s team used microfluidics to determine that marine bacteria change direction in a manner “completely differently than E. coli does” [2].
Swimming or crawling?
Much of the microfluidic chemotactic literature is devoted to studying mammalian phenomena such as cancer, immune and stem cell migration. Although many of the concepts translate to bacteria, not all do. “The main difference is that bacteria [are] in suspension, they are not really crawling on the surface or in a matrix,” explains Francis Lin, who runs the immunotrafficking lab at the University of Manitoba. “The other difference is in the size of the cells.” Bacteria sense the gradient as they swim around, adjusting their movement accordingly, whereas the larger cells tend to determine the gradient first and then migrate by crawling around in it.
For attached cells, it’s very easy to impose a gradient, or to change the chemical environment over time, by simply flowing fluid with different molecules over them, says Stocker. The cells remain in the field of observation. The situation is completely different if you have bacteria, protists, phytoplankton or anything else that lives suspended in the fluid. “If you flush in a different fluid in your microfluidic device, you will flush out the organisms.”
Go with(out) the flow
For many researchers, the solution is to allow diffusion, rather than flow, to establish a gradient [3].
Many different designs have been conceived to enable cells to experience a steady chemotactic gradient in microfluidic device, free from flow that may sweep away secreted biomolecules and from shear forces that could interfere with migration. They typically rely on the ability of the dissolved chemical or chemicals to diffuse from an area of higher concentration to an area of lower concentration, in a way that does not allow bulk flow. This is accomplished by placing a barrier, such as membrane, hydrogel (like agarose) or nanoscale channels, between the two pools.
“At the risk of oversimplifying, they all serve the same purpose: To separate the body of fluid in which you have the cells from the reservoir from which you create diffusion,” says Stocker. “Fluid flow can’t go in, bacteria cannot swim out, but the dissolved molecules can freely traverse that.”
For short-term experiments, setups can be as simple as an etched nitrocellulose membrane sandwiched between glass slides with holes for inlets and outlets. For longer-term investigations, larger reservoirs or continuous flow (driven through tubing by a syringe pump, perhaps) are needed to maintain the chemoeffector concentrations. By manipulating parameters such as the shape and topography of the channel containing the bacteria, the concentrations of chemicals in the reservoirs, the number of opposing or orthogonal chemoeffectors and the distance between the reservoirs, researchers can test a wide variety of conditions.
Flow in the cell chamber is not always a bad thing, either. The gradients can be easier to manipulate, faster to set up and stabilize and can mimic aspects of natural environment such as are found in ground water or the human digestive system. Arul Jayaraman’s μFlow system, for example, allows liquid from separate channels to repeatedly mix and split until a stream emerges that may be of any desired strength and concentration, with a stable gradient generated within five to 10 minutes [4]. He uses this system to query highly motile lab-strain bacteria. “I don’t care if I wash out 90%, because I get enough reliable data in the 10% that registers,” says Jayaraman, professor of chemical engineering at Texas A&M University.
Off the shelf?
Although there are a few products on the market from companies such as BellBrook Labs and ibidi, commercialization of microfluidic chemotactic assay devices has not yet taken off [1]. Instead, researchers will typically use their own design or one developed in another scientific lab and use it with off-the-shelf components like pumps and tubing (if required). The devices can be manufactured by a scattering of facilities, such as the Microtechnology Medicine Biology Lab Foundry (MMB) at the University of Wisconsin (UW) and Dolomite Microfluidics, notes Erwin Berthier, a research scientist at UW’s MMB Lab.
Commercial or DIY, flow-based or static, the advantage of microfluidic devices over traditional, larger-scale devices is that you have a big toolbox, affording greater control and flexibility in setting up gradients, greater sensitivity and statistical power in the experiments and the ability to observe behavior on both the individual and population scales. Who says great things don’t come in small packages?
References
[1] Sackmann, EK, et al., “The present and future role of microfluidics in biomedical research,” Nature, 507:181–9, 2014. [PMID: 24622198]
[2] Son, K, et al., “Bacteria can exploit a flagellar buckling instability to change direction,” Nat Physics, 9:494–8, 2013. [Article link]
[3] Ahmed, T, et al., “Microfluidics for bacterial chemotaxis,” Integr Biol, 2:604–29, 2010. [PMID: 20967322]
[4] Englert, DL, et al., “Flow-based microfluidic device for quantifying bacterial chemotaxis in stable, competing gradients,” Appl Environ Microbiol, 75:4557–64, 2009. [PMID: 19411425]
Image: The ibidi μ-Slide Chemotaxis3D.