Droplet microfluidics has rapidly emerged as one of the key technologies opening up new experimental possibilities in microbiology. The ability to generate, manipulate and monitor droplets carrying single cells or small populations of bacteria in a highly parallel and high throughput manner creates new approaches for solving problems in diagnostics and for research on bacterial evolution. This review presents applications of droplet microfluidics in various fields of microbiology: i) detection and identification of pathogens, ii) antibiotic susceptibility testing, iii) studies of microbial physiology and iv) biotechnological selection and improvement of strains. We also list the challenges in the dynamically developing field and new potential uses of droplets in microbiology.

We report an automated microfluidic platform for 'digitally' screening the composition space of droplets containing cocktails of small molecules and demonstrate the features of this system by studying epistatic interactions between antibiotics and Escherichia coli ATCC 25922. This system has several key characteristics: (i) it uses small (<100 μL) samples of liquids and suspensions of bacteria that are introduced directly into the chip; (ii) it generates a sequence of droplets with compositions, including reagents and bacterial cell suspensions that are programmed by the user; (iii) it exports the sequence of droplets to an external segment of tubing that is subsequently disconnected for incubation and storage; and (iv) after incubation of bacteria in droplets, the droplets are injected into a second device equipped with an in-line fiber optic spectrophotometer that measures cell growth. The system generates and fuses droplets with precise (<1% in standard deviation) control of liquid volumes and of the concentrations of input substrates. We demonstrate the application of this technology in determining the minimum inhibitory concentration and pair-wise interactions of ampicillin, tetracycline, and chloramphenicol against E. coli. The experiments consumed small volumes of reagents and required minutes to create the droplets and several hours for their incubation and analysis.

Droplet microfluidics is a relatively new and rapidly evolving field of science focused on studying the hydrodynamics and properties of biphasic flows at the microscale, and on the development of systems for practical applications in chemistry, biology and materials science. Microdroplets present several unique characteristics of interest to a broader research community. The main distinguishing features include (i) large numbers of isolated compartments of tiny volumes that are ideal for single cell or single molecule assays, (ii) rapid mixing and negligible thermal inertia that all provide excellent control over reaction conditions, and (iii) the presence of two immiscible liquids and the interface between them that enables new or exotic processes (the synthesis of new functional materials and structures that are otherwise difficult to obtain, studies of the functions and properties of lipid and polymer membranes and execution of reactions at liquid-liquid interfaces). The most frequent application of droplet microfluidics relies on the generation of large numbers of compartments either for ultrahigh throughput screens or for the synthesis of functional materials composed of millions of droplets or particles. Droplet microfluidics has already evolved into a complex field. In this review we focus on 'controlled droplet microfluidics' - a portfolio of techniques that provide convenient platforms for multistep complex reaction protocols and that take advantage of automated and passive methods of fluid handling on a chip. 'Controlled droplet microfluidics' can be regarded as a group of methods capable of addressing and manipulating droplets in series. The functionality and complexity of controlled droplet microfluidic systems can be positioned between digital microfluidics (DMF) addressing each droplet individually using 2D arrays of electrodes and ultrahigh throughput droplet microfluidics focused on the generation of hundreds of thousands or even millions of picoliter droplets that cannot be individually addressed by their location on a chip.

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This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated.

We separate emulsions with an immiscible oil phase to identify reaction conditions by the location of emulsion in emulsion series.

We describe microfluidic technology for manipulating and monitoring continuous growth of populations of bacteria. A system consisting of ∼10 input and output channels controls >10 2 microdroplet chemostats and enables the manipulation of chemical factors in each microchemostat independently over time. This paper characterizes the dynamics of bacterial populations in microdroplet chemostats and cellular responses to a range of stable or changing antibiotic concentrations. This technology provides a platform for highly parallel, long-term studies of microbial ecology, physiology, evolution, and adaptation to chemical environments.The introduction of the chemostat by Leo Szilard [1] was a milestone in the field of microbiology. Chemostats facilitate the continuous culture of bacteria, yeast, and algae by continuously replenishing a constant volume of fluid to maintain specific concentrations of cells and growth factors. [1,2] Chemostats have facilitated a wide-range of studies, including microbial ecology, [3] predator-prey dynamics [4] and the evolution of drug resistance [5,6] The ** This project was performed co-financed by the EU European Regional Development Fund under the Operational Programme Innovative Economy NanoFun POIG.02.02.00-00-025/09 (to P.G.) and within the European Research Council Starting Grant 279647 (to P.G.).Correspondence to: Piotr Garstecki, garst@ichf.edu.pl. + These authors contributed equally to this work.Supplemental Information including experimental details, is available on the WWW under …. consumption of large quantities of reagents and significant operational challenges of traditional chemostats limit their use. NIH Public AccessSingle phase, microfluidic versions of chemostats minimize incubation volumes, [7][8][9][10] and yet are limited by their complexity: the proportionality between the number of input/output controls and the number of chemostats hamper large scale parallelization Single-phase microfluidic systems are prone to biofilm formation, which makes them either single-use devices [9] or requiring additional steps to minimize cell adhesion. [8] Droplet microfluidics [11] offers a unique solution to creating many parallel chemostats. The earliest example of this technology in microbiology was first demonstrated by Joshua Lederberg nearly 60 years ago. [12] In the interim, the field of microfluidics solved many of the technical challenges associated with using this approach to study microbes.Compartmentalizing cells and nutrients in microdroplets of liquid can reduce the complexity and cost of operating many parallel chemostats. Recently, bacteria have been incubated in droplets in channels over short time intervals, [13][14][15][16][17] however sustained cell growth over hundreds of generations in a series of fully addressable microdroplets has not been possible.Here, we describe an automated microdroplet system that transcends existing challenges and enables users to manipulate the chemical composition of droplets for long-term bacterial studies. The microfluidic system ...

This paper proves that dodecylresorufin (C12R) outperforms resorufin (the conventional form of this dye) in droplet microfluidic bacterial assays. Resorufin is a marker dye that is widely used in different fields of microbiology and has increasingly been applied in droplet microfluidic assays and experiments. The main concern associated with resorufin in droplet-based systems is dye leakage into the oil phase and neighboring droplets. The leakage decreases the performance of assays because it causes averaging of the signal between the positive (bacteria-containing) and negative (empty) droplets. Here we show that C12R is a promising alternative to conventional resorufin because it maintains higher sensitivity, specificity, and signal-to-noise ratio over time. These characteristics make C12R a suitable reagent for droplet digital assays and for monitoring of microbial growth in droplets.