Droplet Microfluidics for Quantifying Phage-Host Dynamics

Bacteriophages, or phages, are viruses that infect bacteria. They have shaped molecular biology for decades and are gaining renewed attention. However, studying how phages infect bacteria comes with technical challenges. The classic double-layer plaque assay has long been the standard method for phage quantification, but it is slow, fixed in format, and poorly suited for observing infection dynamics over time. It measures the outcome of many infection cycles rather than the behavior of individual phage-host encounters. In a recent article in Nature Communications, Givelet and colleagues address this limitation by developing a high-throughput droplet microfluidic platform that can measure individual phage infection events in thousands to hundreds of thousands of isolated droplets.

Their proposed solution is a digital phage assay based on droplet microfluidics. Instead of mixing phages and bacteria in bulk before analysis, the researchers co-encapsulated them into water-in-oil droplets using a microfluidic device. Each droplet acts as an isolated reaction chamber where phage exposure begins at the moment of encapsulation. This design makes it possible to control exposure time, droplet volume, and the ratio of phages to bacteria. It also prevents newly produced phages from spreading into the rest of the sample, which is a major problem in bulk liquid culture assays. As a result, the method can focus on discrete lysis events caused by the initially encapsulated phages.

The microfluidic chip was mirofabricated using PDMS and standard lithography microfluidic fabrication techniques. During experiments, separate aqueous streams containing bacteria and phages were introduced into a flow-focusing microfluidic chip, while the oil with fluorinated surfactant formed the continuous phase. This dual co-flow design ensured that bacteria and phages met only at the droplet generation junction, giving the researchers better control over the start of infection.

To detect lysis, the researchers used YOYO-1, a cell-impermeable fluorescent DNA dye. When a bacterium lyses, its DNA is released into the droplet and binds the dye, producing a stronger green fluorescence signal. They also used a red fluorescent reference dye, Atto 565, to obtain information about droplet size and the mixing fraction between the phage and bacterial suspensions. After droplet generation, the emulsions were incubated at 37 °C and then reinjected into a separate microfluidic screening chip. A high-speed fluorescence detection setup scanned droplets at rates up to several kilohertz, collecting red and green fluorescence signals from each droplet. T

One important part of the method is the statistical model used to convert droplet fluorescence data into phage titers. The authors assumed that a droplet becomes positive when it contains at least one bacterium and at least one infective phage. Using Poisson statistics, they related the fraction of positive droplets to the expected number of encapsulated bacteria and phages. A Gaussian mixture model was then used to classify droplets into positive and negative populations based on green fluorescence intensity. This avoided relying on a fixed fluorescence threshold and helped reduce user bias during analysis.

The results showed that the droplet microfluidic assay could produce digital phage titers that closely matched values obtained by the traditional double-layer plaque assay. These results suggest that droplet microfluidics can serve as a quantitative alternative to plaque-based phage enumeration while also giving access to more detailed infection information.

The authors also showed that changing the mixing fraction between phage and bacterial suspensions can expand the dynamic range of the assay. By adjusting the pressures at the two aqueous inlets, they generated droplet subpopulations with different phage-to-bacteria ratios while keeping droplet size relatively constant. This allowed them to examine how the fraction of positive droplets changed as the amount of phage suspension in each droplet varied. Interestingly, the observed results did not always match the simplest theoretical expectation, which led the authors to consider the role of effective bacterial density. Not every cell counted by optical density is necessarily active, susceptible, or capable of supporting phage infection. The droplet microfluidic platform therefore provided a way to estimate not just phage titer, but also the effective population of bacteria participating in lysis.

In conclusion, this study presents a powerful droplet microfluidic device for quantifying phage-host dynamics at the level of individual infection events. By isolating phages and bacteria in droplets, the platform avoids the confounding effects of repeated progeny-driven infections in bulk culture. It can measure phage titers, estimate effective host participation, test different mixing ratios, and track lysis kinetics over time. The approach could support fundamental phage biology, phage therapy research, and future high-throughput screening of phage variants with desirable infection properties.

Figures are reproduced from Givelet, L., von Schönberg, S., Katzmeier, F. et al. Quantifying phage-host dynamics using droplet microfluidics. Nat Commun 17, 3857 (2026). https://doi.org/10.1038/s41467-026-72427-3 under a Creative Commons Attribution 4.0 International License.

Read the original article: Quantifying phage-host dynamics using droplet microfluidics

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