title: Rapid enumeration of phage in monodisperse emulsions.
abstract: Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.
authors.affiliation: Department of Chemistry, University of Alberta , Edmonton, AB T6G 2G2, Canada.
authors.forename: Katrina F
authors.initials: KF
authors.lastname: Tjhung
authors.forename: Sean
authors.initials: S
authors.lastname: Burnham
authors.forename: Hany
authors.initials: H
authors.lastname: Anany
authors.forename: Mansel W
authors.initials: MW
authors.lastname: Griffiths
authors.forename: Ratmir
authors.initials: R
authors.lastname: Derda
mesh_terms: D017104:Bacteriophage M13 / Q000302:isolation & purification*; D015169:Colony Count, Microbial; D004655:Emulsions
chemical_list: D004655:Emulsions