Automated Chemotactic Sorting and Single-cell Cultivation of Microbes using Droplet Microfluidics

Abstract

We report a microfluidic device for automated sorting and cultivation of chemotactic microbes from pure cultures or mixtures. The device consists of two parts: in the first part, a concentration gradient of the chemoeffector was built across the channel for inducing chemotaxis of motile cells; in the second part, chemotactic cells from the sample were separated, and mixed with culture media to form nanoliter droplets for encapsulation, cultivation, enumeration, and recovery of single cells. Chemotactic responses were assessed by imaging and statistical analysis of droplets based on Poisson distribution. An automated procedure was developed for rapid enumeration of droplets with cell growth, following with scale-up cultivation on agar plates. The performance of the device was evaluated by the chemotaxis assays of Escherichia coli (E. coli) RP437 and E. coli RP1616. Moreover, enrichment and isolation of non-labelled Comamonas testosteroni CNB-1 from its 1:10 mixture with E. coli RP437 was demonstrated. The enrichment factor reached 36.7 for CNB-1, based on its distinctive chemotaxis toward 4-hydroxybenzoic acid. We believe that this device can be widely used in chemotaxis studies without necessarily relying on fluorescent labelling, and isolation of functional microbial species from various environments.

Figure 1

(A) Schematic of the device for chemotactic sorting and droplet cultivation; (B) Photograph of a device filled with food dye solutions. (C) Zoom-in view of food dye droplet generation on the device.

Figure 2. Chemotaxis assays based on parallel-flow.

(A) Numerical simulation of diffusion gradient at the interface of parallel-flow in the main channel, assuming a total flow rate of 0.53 μL/min. (B,C) Fluorescent imaging of the diffusion gradients for fluorescein in the main channel, at the inlet and 4 mm downstream from the inlet. (D) Experimental validation of cross-sectional diffusion profiles at 0 mm (red line) and 4 mm (blue line) below the junction were compared with theoretical simulations. (E) Representative fluorescent image showing response of E. coli RP437 to the gradient of 10 mM aspartic acid (Asp). (F) Zoom-in view shows that some cells were not clearly imaged (indicated by the white arrows). The experiments were performed at a total flow rate of 0.53 μL/min.

Figure 3. Evaluation of chemotaxis of microbial cells based on nanoliter droplet cultivation.

(A) Percentages of cell-containing droplets in array for serial diluted cell suspensions. E. coli RP437 was used as the sample to direct mix with culture media to form droplets. The experimental data is in good agreement with theoretical predication (black dotted curve). (B) Difference of the average number of cells per droplet (λ) for E. coli RP437 and E. coli RP1616 with and without 10 mM Asp as the chemoeffector. A 1:1 mixture of E. coli RP437 and E. coli RP1616 was used as the sample.

Figure 4. Rapid enumeration of droplets with cell growth.

(A) Schematic of automated droplet array detection and scale-up cultivation of the droplet array. (B,C) Typical bright-field microscopic images of empty droplets (B) and cell-containing droplets (C). E. coli RP437 was used as the cell sample. Scale bar = 50 μm. (D) Colony growth of E. coli RP437 on agar plate inoculated with a single droplet. (E) Distinguish empty and cell-containing droplets using bright-field (BF) video recording. (F) Box plot of BF intensities for empty droplets and cell-containing droplets.

Figure 5. Separation of non-labelled chemotactic cells from mixtures.

(A) Calibration of chemotaxis of non-labelled species with a fluorescent labelled reference strain. Droplets are imaged using both bright-field and fluorescence microscopy, and classified as empty, only reference strain, only non-labelled strain and both strain. The cell density of non-labelled strain was analyzed based on Poisson distribution. (B,C) Phase-contrast microscopic images of droplets in Teflon tubing after 24 h cultivation: (B) empty, (C) with growth of non-labelled CNB-1. Scale bar = 25 μm. (D) Evaluation of the chemotaxis of non-labelled CNB-1 using droplet-based enumeration, comparing before and after exposure to p-HBA with the same experimental setting. (E) Chemotactic sorting of non-labelled CNB-1 from its 1:10 mixture with E. coli RP437, comparing before and after exposure to p-HBA.