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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Environment

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Published: May 5th, 2020

DOI:

10.3791/61062

1Institute of Environmental Engineering, Department of Civil, Environmental and Geomatic Engineering, ETH Zürich, 2Climate Change Cluster, University of Technology Sydney, 3School of Oceanography, University of Washington

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 stud....

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We recommend executing section 1 prior to field experiments to optimize results.

1. Laboratory optimization

NOTE: The volumes described in the optimization procedure are sufficient for a single ISCA (composed of 20 wells).

  1. Preparation of the chemical of interest
    NOTE: The optimal concentration for each chemoattractant often needs to be determined under laboratory conditions prior to field deployments. The chemical concentration field will decrease.......

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This section presents laboratory results using the ISCA to test the chemotactic response of marine microbes to a concentration range of glutamine, an amino acid known to attract soil bacteria14. The concentration of glutamine that elicited the strongest chemotactic response in the laboratory tests was used to perform a chemotaxis assay in the marine environment.

To perform the laboratory tests, seawater communities sampled from coastal water in Sydney, Australia, were e.......

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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.......

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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.

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Name Company Catalog Number Comments
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

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