Continuous-flow capillary assay for measuring bacterial chemotaxis

Abstract

Bacterial chemotaxis may have a significant impact on the structure and function of bacterial communities. Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the bacterial transport parameters used in predictive models of chemotaxis. When the chemotactic bacteria consume the chemoeffector, the chemoeffector gradient to which the bacteria respond may be significantly perturbed by the consumption. Therefore, consumption of the chemoeffector can confound chemotaxis measurements if it is not accounted for. Current methods of quantifying chemotaxis use bacterial concentrations that are too high to preclude chemoeffector consumption or involve ill-defined conditions that make quantifying chemotaxis difficult. We developed a method of quantifying bacterial chemotaxis at low cell concentrations ( approximately 10(5) CFU/ml), so metabolism of the chemoeffector is minimized. The method facilitates quantification of bacterial-transport parameters by providing well-defined boundary conditions and can be used with volatile and semivolatile chemoeffectors.

FIG. 1.

Diagram (I) and photograph (II) of the continuous-flow capillary assay apparatus. Bacterial suspension is pumped into the flow channel (A) through the stainless-steel block (B), past the capillaries (C), and through an exit port (D). The stainless-steel block is a cylinder with a diameter of 38 mm and a height of 50 mm. The cylindrical flow channel through the center is 6 mm in diameter, and the capillary ports are located 38 mm from the bottom of the stainless-steel block.

FIG. 2.

Photograph and schematic of capillary assembly showing the stainless-steel plunger (A), septum (B), sheath (C), HPLC fitting that holds the capillary and screws into the steel block (D), viton o-ring (E), and open mouth of the capillary that is inserted into the flowing bacterial suspension (F). The outside and inside diameters of the capillary are 1.0 and 0.5 mm, respectively. In the photograph, the sheath is omitted for clarity and the plunger is shown pushed all the way to the mouth of the capillary.

FIG. 3.

Mass of salicylate that diffused into or out of capillaries versus time. The data comprise measurements of the mass of salicylate exiting the capillaries (▪) and entering the water flowing through the assay apparatus flow channel (⋄), or entering the capillaries (▴) from the flowing water. The solid line is equation 1 plotted using the theoretical value for the diffusion coefficient, 8.8 × 10⁻⁶ cm²/s. The error bars are 1 standard deviation of three independent measurements. The initial salicylate concentration was 25 mM.

FIG. 4.

Accumulation of Pseudomonas putida G7 cells in capillaries by chemotaxis to naphthalene (▴) or random motility (⋄). The initial bacterial concentration was 4 × 10⁵ CFU/ml. The solid line is a solution to the bacterial-transport equation using the fitted random-motility coefficient, 3.0 × 10⁻⁶ cm²/s, and the dashed line is a solution to the equation using the fitted random-motility coefficient and the fitted chemotactic-sensitivity coefficient, 6.7 × 10⁻⁵ cm²/s. The error bars are 1 standard deviation of measurements from three capillaries. Naphthalene-saturated motility buffer was used in the capillaries for the chemotaxis experiment.

FIG. 5.

Effect of flow rate on bacterial accumulation in capillaries initially filled with naphthalene-saturated motility buffer. Bacterial accumulations were measured at 35 min, and the initial bacterial concentration was 10⁶ CFU/ml. The error bars are the standard deviation from five replicate measurements.