Micro-confinement of bacteria into w/o emulsion droplets for rapid detection and enumeration

Abstract

Single-cell characterisation and rapid enumeration of E. coli was achieved by confining them into the picoliter droplets of a water-in-oil fuorinated emulsion. Micro-confinement of Bacteria into w/o Emulsion Droplets for Rapid Detection and Enumeration AbstractToday, rapid detection and identification of bacteria in microbiological diagnosis is a major issue. Reference methods usually rely on growth of micro-organisms, with the drawback of lengthy time-to-result. The method provides global information on a clonal population that is known to be inhomogeneous relative to metabolic states and activities. Therefore, there may be a significant advantage of methods that allow characterization of individual bacteria from a large population, both for test time reduction and the clinical value of the characterization. We report here a method for rapid detection and real-time monitoring of the metabolic activities of single bacteria. Water-in-oil emulsions were used to encapsulate single Escherichia coli cells into picolitre (pL)-sized microreactor droplets. The glucuronidase activity in each droplet was monitored using the fluorogenic reporter molecule MUG (4-Methylumbelliferyl β-D-glucuronide) coupled to time-lapse fluorescence imaging of the droplets. Such bacterial confinement provides several major advantages. 2 1) Enzymatic activities of a large number of single bacterium-containing droplet could be monitored simultaneously, allowing the full characterization of metabolic heterogeneity in a clonal population. We monitored glucuronidase enzymatic activity and growth over ~200 single bacteria over a 24h-period. 2) Micro-confinement of cells in small volumes allows rapid accumulation of the fluorescent metabolite, hence decreasing the detection time. Independent of the initial concentration of bacteria in the sample, detection of the presence of bacteria could be achieved in less than two hours. 3) Considering the random distribution of bacteria in droplets, this method allowed rapid and reliable enumeration of bacteria in the initial sample. Overall, the results of this study showed that confinement of bacterial cells increased the effective concentration of fluorescent metabolites leading to rapid (2 h) detection of the fluorescent metabolites, thus significantly reducing time to numeration.

Uniform amplification of phage display libraries in monodisperse emulsions

“…It is important to prevent coalescence of droplets during the incubation period. In previous reports, coalescence of droplets was prevented by storing the droplets inside gas permeable tubing [15,16,30]. Here we use a surfactant, developed by Weitz and co-workers [13], which equips droplets with exceptional stability.…”

“…For example, Ismagilov and co-workers [30], stabilized aqueous droplets by using a triethyleneglycolmonol[1H,1H-perfluorooctyl]ether (RfOEG) surfactant, and used these droplets to culture rare bacteria. Due to the short length of the fluorinated chain in RfOEG, the droplets stabilized by this surfactant are prone to coalescence on contact [16]. To avoid coalescence, the bacteria could be cultured as a sequence of drops inside gas permeable tubing.…”

“…These droplets can serve as compartments for growth of cells and bacteria [13,15,16]. Droplet size is the function of channel geometry and pressure in the channel; if pressure is constant, the droplets produced in the channel have nearly uniform size (polydispersity of 1.01) [17,18].…”

Miniaturized Detection Systems

“…81 Detection was based on metabolism, showing good agreement with traditional colony counts, achievable within 2 h for some bacteria, with full quantification possible in less than 10 h (Fig. 10.5).…”

Quantitative detection of cells expressing BlaC using droplet-based microfluidics for use in the diagnosis of tuberculosis

“…To our knowledge, no prior work has applied droplet microfluidics for the detection of TB. [18][19][20][21][22] The bacterial sample is prepared at a limiting dilution such that each drop contains one or no cell. We use Escherichia coli (E. coli) expressing BlaC as a surrogate to characterize our method and validate some of our results using Bacillus Calmette-Gu erin (BCG), a strain of attenuated Mycobacterium bovis.…”