Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.
Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.
Diverse microorganisms use motility and chemotaxis to exploit patchy nutrient environments, find hosts, or avoid deleterious conditions1,2,3. These microbial behaviours can in turn influence rates of chemical transformation4 and promote symbiotic partnerships across terrestrial, freshwater, and marine ecosystems2,5.
Chemotaxis has been extensively studied under laboratory conditions for the past 60 years6. The first quantitative method to study chemotaxis, the capillary assay, involves a capillary tube filled with a putative chemoattractant immersed in a suspension of bacteria6. Diffusion of the chemical out of the tube creates a chemical gradient, and chemotactic bacteria respond to this gradient by migrating into the tube7. Since the development of the capillary assay, still widely used today, many other techniques have been developed to study chemotaxis under increasingly controlled physical/chemical conditions, with the most recent involving the use of microfluidics8,9,10.
Microfluidics, together with high-speed video microscopy, enables tracking of the behavior of single cells in response to carefully controlled gradients. Although these techniques have vastly improved our understanding of chemotaxis, they have been restricted to laboratory use and do not translate easily to field deployment in environmental systems. As a consequence, the capacity of natural communities of bacteria to use chemotaxis within natural ecosystems has not been examined; thus, current understanding of the potential ecological importance of chemotaxis is biased toward artificial laboratory conditions and a limited number of laboratory-cultured bacterial isolates. The recently developed ISCA overcomes these limitations11.
The ISCA builds on the general principle of the capillary assay; however, it makes use of modern microfabrication techniques to deliver a highly replicated, easily deployable experimental platform for the quantification of chemotaxis toward compounds of interest in the natural environment. It also allows identification and characterization of chemotactic microorganisms by direct isolation or molecular techniques. While the first working device was self-fabricated and constructed of glass and PDMS11, the latest injection-molded version is composed of polycarbonate, using a highly standardized fabrication procedure (for interest in the latest version of the device, the corresponding authors can be contacted).
The ISCA is credit card-sized and consists of 20 wells distributed in a 5 x 4 well array, each linked to the external aquatic environment by a small port (800 µm in diameter; Figure 1). Putative chemoattractants loaded into the wells diffuse into the environment via the port, and chemotactic microbes respond by swimming through the port into the well. As many factors can influence the outcome of an ISCA experiment in the natural environment, this step-by-step protocol will help new users overcome potential hurdles and facilitate effective deployments.
At the scale of aquatic microorganisms, the environment is far from homogenous and is often characterized by physical/chemical gradients that structure microbial communities1,15. The capacity of motile microorganisms to use behavior (i.e., chemotaxis) facilitates foraging within these heterogeneous microenvironments1. Studying chemotaxis directly in the environment has the potential to identify important interspecific interactions and chem…
The authors have nothing to disclose.
This research was funded in part by the Gordon and Betty Moore Foundation Marine Microbiology Initiative, through grant GBMF3801 to J.R.S. and R.S., and an Investigator Award (GBMF3783) to R.S., as well as an Australian Research Council Fellowship (DE160100636) to J.B.R., an award from the Simons Foundation to B.S.L. (594111), and a grant from the Simons Foundation (542395) to R.S. as part of the Principles of Microbial Ecosystems (PriME) Collaborative.
Acrylic glue | Evonik | 1133 | Acrifix 1S 0116 |
Acrylic sheet | McMaster-Carr | 8505K725 | Or different company |
Adhesive tape | Scotch | 3M 810 | Scotch Magic tape |
Autoclave | Systec | D-200 | Or different company |
Benchtop centrifuge | Fisher Scientific | 75002451 | Or different company |
Bungee cord | Paracord Planet | 667569184000 | Or different company |
Centrifuge tube – 2 mL | Sigma Aldrich | BR780546-500EA | Eppendorf tube |
Conical centrifuge tube – 15 mL | Fisher Scientific | 11507411 | Falcon tube |
Conical centrifuge tube – 50 mL | Fisher Scientific | 10788561 | Falcon tube |
Deployment arm | Irwin | 1964719 | Or different company |
Deployment enclosure plug | Fisher Scientific | 21-236-4 | See alternatives in manuscript |
Disposable wipers | Kimtech – Fisher Scientific | 06-666 | Kimwipes |
Flow cytometer | Beckman | C09756 | CYTOFlex |
Glutaraldehyde 25% | Sigma Aldrich | G5882 | Or different company |
Green fluorescent dye | Sigma Aldrich | S9430 | SYBR Green I – 1:10,000 final dilution |
Hydrophilic GP filter cartridge – 0.2 µm | Merck | C3235 | Sterivex filter |
In Situ Chemotaxis Assay (ISCA) | – | – | Contact corresponding authors |
Laser cutter | Epilog Laser | Fusion pro 32 | Or different company |
Luria Bertani Broth | Sigma Aldrich | L3022 | Or different company |
Marine Broth 2216 | VWR | 90004-006 | Difco |
Nylon slotted flat head screws | McMaster-Carr | 92929A243 | M 2 × 4 × 8 mm |
Pipette set | Fisher Scientific | 05-403-151 | Or different company |
Pipette tips – 1 mL | Fisher Scientific | 21-236-2A | Or different company |
Pipette tips – 20 µL | Fisher Scientific | 21-236-4 | Or different company |
Pipette tips – 200 µL | Fisher Scientific | 21-236-1 | Or different company |
Sea salt | Sigma Aldrich | S9883 | For artificial seawater |
Serological pipette – 50 mL | Sigma Aldrich | SIAL1490-100EA | Or different company |
Syringe filter – 0.02 µm | Whatman | WHA68091002 | Anatop filter |
Syringe filter – 0.2 µm | Fisher Scientific | 10695211 | Or different company |
Syringe needle 27G | Henke Sass Wolf | 4710004020 | 0.4 × 12 mm |
Syringes – 1 mL | Codau | 329650 | Insulin Luer U-100 |
Syringes – 10 mL | BD | 303134 | Or different company |
Syringes – 50 mL | BD | 15899152 | Or different company |
Tube rack – 15 mL | Thomas Scientific | 1159V80 | Or different company |
Tube rack – 50 mL | Thomas Scientific | 1159V80 | Or different company |
Uncoated High-Speed Steel General Purpose Tap | McMaster-Carr | 8305A77 | Or different company |
Vacuum filter – 0.2 µm | Merck | SCGPS05RE | Steritop filter |