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. 2020 Jun 17;9:e56998. doi: 10.7554/eLife.56998

Figure 1. End-to-end system for efficient isolation and culture of gut anaerobes in microfluidic droplets.

(a) The experimental setup for isolating, culturing, and sorting anaerobic bacteria in microfluidic droplets consists of a microscope, microfluidic devices, a high frame rate camera, syringe pumps, an incubator, and electrodes all contained within an anaerobic chamber. The computer controls the syringe pumps, high frame rate camera, and electrodes (via a voltage amplifier). The equipment power and control wires are introduced to the anaerobic chamber through sealed rubber ports to strictly maintain the anaerobic conditions within the chamber. (b) Single bacteria cells are isolated in droplets containing anaerobic culture medium and the resulting emulsion is cultured inside the incubator. (c) An example of human gut bacteria isolated and cultivated inside droplets. (d) Droplets are sorted by optical detection and subsequent deflection via dielectrophoresis near a sorting junction. Specifically, droplets with bacterial colonies which meet a certain thresholding criteria were determined using image analysis (region between the red dots), and these droplets were deflected into the ‘keep’ path by actuating an on-chip electrode while sending the remaining droplets to waste. (e) The colony density measured by image analysis (Wavelet OD) for 1000 successive droplets. Droplets with a dense colony, a sparse colony, and empty droplets (no colony) are represented by a wavelet OD value above an upper threshold, between an upper and lower threshold, or below a lower threshold, respectively. (f) Two slow-growing human gut-associated bacteria colonies (bottom left and bottom right) grown in droplets after sorting and a false positive empty droplet (top). Scale bar in (b) and (d) is 100 μm and scale bar in (c) and (f) is 20 μm.

Figure 1.

Figure 1—figure supplement 1. The Altered Schaedler’s Flora, ASF, is an important and widely studied gnotobiotic mouse model used for understanding microbiota-host dynamics in both health and disease.

Figure 1—figure supplement 1.

The ASF community contains two facultative anaerobes, Lactobacillus intestinalis (ASF 360) and Lactobacillus murinus (ASF 361), two anaerobes, Mucispirillum schaedleri (ASF 457) and Parabacteroides goldsteinii (ASF 519), and four extremely oxygen sensitive anaerobes, Clostridium sp. (ASF 356), Eubacterium plexicaudatum (ASF 492), Pseudoflavonifactor sp. (ASF 500), and Clostridium sp. (ASF 502) (Wymore Brand et al., 2015). Pure ASF strains were cultured from stock solutions in liquid BHIS-ASF broth, then diluted and encapsulated in droplets using our microfluidic platform, and incubated at 37°C. After 17 to 20 hr, we observed that all 8 ASF strains were successfully cultured in the droplets. Scale bars are 20 μm.
Figure 1—figure supplement 2. Droplet volume decreased during cultivation in the anaerobic chamber.

Figure 1—figure supplement 2.

The figure shows cultivation of the donor FMT sample in BHIS droplets at 1 day, 2 days, and 4 days of cultivation time. The droplet volume decreased ~10 x from Day 1 to Day 4.