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.
Our protocol enables us to robustly quantify microbial chemotaxis directly in their home habitat. And thereby we can isolate microorganisms and we can better quantify and understand the metabolic potential. The in situ chemotaxis assay, or ISCA in short, is a flexible, user-friendly, and simple device that allows new insight into microbial behaviors and therefore the role and function of microorganisms in any environment that has a liquid phase.
Demonstrating the procedure for deployment in both the laboratory and in the field is Estelle Clerc, who is currently a PhD student in my research group. Before preparing the chemoattractant, filter the medium with a 0.2 micrometer filter, and autoclave the filtered solution. Next, prepare a 10 millimolar solution of chemoattractant in 1 milliliter of the sterile medium, and filter the chemoattractant solution with a 0.2 micrometer syringe filter to remove any particles and potential contaminants.
Then, dilute the filtered chemoattractant stock solution in series, according to the experimental protocol. To fill an ISCA, attach a 27 gauge needle to a 1 milliliter syringe, and load one syringe for each concentration of the filtered chemoattractant. Holding the device with the port facing upwards, slowly inject each concentration of the substance into each of the five wells of one row of the device until a small droplet appears on top of each port.
When all of the wells have been filled, transfer 1.5 milliliter of the marine or freshwater bacteria culture of interest in 150 milliliters of the appropriate bacterial culture medium. Next, place two small pieces of double-sided adhesive tape on the flat surface of a 200 milliliter capacity tray. And secure one ISCA onto each piece of tape.
Using a 50 milliliter serological pipette, slowly fill the deployment tray with the bacterial solution. And allow the bacteria to respond to the chemoattractant of interest for one hour. To end the analysis, use a new 50 milliliter pipette to gently remove the medium from the ISCA tray.
To retrieve the samples, first position the ISCA so that the port faces downward. Next, use a sterile 1 milliliter syringe equipped with a 27 gauge needle to draw the solution from each well. Pulling the samples from each row of the same concentration into a single tube.
Then, analyze the samples by flow cytometry to determine the number of bacteria attracted to each chemoattractant concentration. To prepare an ISCA for field deployment, first, collect 5 milliliters of water per ISCA from the field side and filter the water through a 0.2 micrometer syringe filter into a 50 milliliter conical tube. Pass the syringe filtered water through a hydrophilic GP filter cartridge two times, collecting the filtrate in a new 50 milliliter conical centrifuge tube, after each rinse.
Then, filter the water through a 0.02 micrometer syringe filter into a new 50 milliliter tube. Use aliquots of the filtrate to resuspend all of the chemoattractants of interest to the desired concentrations in individual 15 milliliter conical tubes. Then use 10 milliliter syringes to filter the resuspended chemoattractants through individual 0.2 micrometer syringe filters into sterile 15 milliliter conical centrifuge tubes to remove any unwanted particles or water insoluble compounds.
For ISCA field deployment, screw each ISCA onto piece 9 of the device enclosure and close the enclosure. Seal the enclosure with adhesive tape and use bungee cords to secure the enclosure to a deployment arm and human made structure. Alternatively, the enclosure can be secured with a small weight on a shallow substrate or attached to a net in the Pelagic ocean.
Submerge the enclosure completely to start the filling, holding the enclosure firmly to prevent excessive water movement inside. Once the level of the water has reached the top of the enclosure, make sure that no air is trapped inside. When the enclosure is completely full, seal the bottom and top holes with two plugs.
Then, leave the ISCA in place for 1 to 3 hours. At the end of the sampling period, remove the enclosure from the water. Place the enclosure over a container to drain out the water, and carefully remove the upper part of the adhesive tape from the front holes.
Once the water line has passed below the top of the ISCA, remove the bottom plug and drain the rest of the water. While the ISCAs are still attached to the enclosure, use a 1 milliliter pipette, or a 1 milliliter syringe, to carefully remove the water trapped on top of each ISCA and remove the ISCAs without touching the upper surface. Then use a disposable wipe to remove any remaining liquid on the surface, and retrieve the samples as demonstrated for the laboratory sample retrieval.
In this representative in vitro analysis, 1 millimolar was determined to be the optimal glutamine deployment concentration as it induced a significant chemotactic response that was 18 fold higher than the filtered seawater control. Higher and lower concentrations of glutamine also induced significant, but weaker, chemotactic response. In this field deployment analysis, five ISCA replicates filled with 1 millimolar glutamine were deployed for 1 hour at a coastal site near Sydney, Australia.
And the glutamine filled ISCA attracted nearly three times more bacteria than the control wells filled with filtered seawater. The most important thing to remember when deploying the ISCA, both in the lab and in the field, is to avoid creating strong fluid flows which will prevent chemotaxis. ISCA derived samples can be used to identify specific organisms or genes, to characterize the genomic potential of chemotactic organisms, and for targeted isolation of specific strains on selective media.
