Enumeration of bacteriophages by double agar overlay plaque assay

Public Health Agency of Canada, Laboratory for Foodborne Diseases, Guelph, Ontario, Canada.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2009; 501:69-76. DOI: 10.1007/978-1-60327-164-6_7
Source: PubMed

ABSTRACT The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. In this chapter the classical overlay protocol is described.

  • Source
    • "A lytic phage (F2047B) (Budinoff, 2012; Ankrah et al., 2014) was isolated from viral concentrates of Raunefjorden sea water using standard bacteriophage enrichment (Van Twest and Kropinski, 2009; Wommack et al., 2009). Plaque purification and preparation of phage stocks were based on previously described methods (Kropinski et al., 2009). Concentrated lysates were made by gently washing soft agar from plaque assay plates with MSB buffer. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Viruses contribute to the mortality of marine microbes, consequentially altering biological species composition and system biogeochemistry. Although it is well established that host cells provide metabolic resources for virus replication, the extent to which infection reshapes host metabolism at a global level and the effect of this alteration on the cellular material released following viral lysis is less understood. To address this knowledge gap, the growth dynamics, metabolism and extracellular lysate of roseophage-infected Sulfitobacter sp. 2047 was studied using a variety of techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. Quantitative estimates of the total amount of carbon and nitrogen sequestered into particulate biomass indicate that phage infection redirects ∼75% of nutrients into virions. Intracellular concentrations for 82 metabolites were measured at seven time points over the infection cycle. By the end of this period, 71% of the detected metabolites were significantly elevated in infected populations, and stable isotope-based flux measurements showed that these cells had elevated metabolic activity. In contrast to simple hypothetical models that assume that extracellular compounds increase because of lysis, a profile of metabolites from infected cultures showed that >70% of the 56 quantified compounds had decreased concentrations in the lysate relative to uninfected controls, suggesting that these small, labile nutrients were being utilized by surviving cells. These results indicate that virus-infected cells are physiologically distinct from their uninfected counterparts, which has implications for microbial community ecology and biogeochemistry.The ISME Journal advance online publication, 5 December 2013; doi:10.1038/ismej.2013.216.
    The ISME Journal 05/2014; 8:1089-1100. DOI:10.1038/ismej.2013.216 · 9.27 Impact Factor
    • "Phage isolates were examined for various physical properties to obtain more distinguishing characteristics. Large plaques of phages can be obtained by using low concentration (0Á3%) of agar (Kropinski et al. 2009). Pseudomonas chlororaphis-specific phages (201U2-1 and 201U2-2), which had different dimensions of about twofold in both head diameter and tail length, were discriminated based on plaque size in low strength gel (0Á1% agarose) (Serwer et al. 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: To isolate and characterise listeriaphages from seafood environments. Listeriaphages (phages) isolated from seafood environments were distinguished by physical and biological techniques including restriction digestion of phage DNA. Three phages belonged to order Caudovirales and showed psychrotrophic characteristics. The phages had broad host ranges against 23 Listeria strains by productive infection or at least by adsorption. At 15 ± 1°C, adsorption rate constants of the three phages ranged from 8.93 x 10(-9) to 3.24 x 10(-11) ml min(-1) across different L. monocytogenes strains. In indicator hosts, the mean burst sizes of phages LiMN4L, LiMN4p and LiMN17 were ≈17, 17 and 11 plaque forming units (PFU) per cell, respectively at 15 ± 1°C. The respective latent periods were ≈270 min for phages LiMN4p and LiMN17 whereas for phage LiMN4L, it was ≈240 min. The three virulent psychrotrophic phages isolated from seafood processing environments had broad host ranges and low productive replication. These characteristics suggest that the phages may be suitable as passive biocontrol agents against seafood-borne L. monocytogenes. This is the first report on isolation of autochthonous virulent listeriaphages from seafood processing environments and information on single-step replication and adsorption characteristics of such listeriaphages. This article is protected by copyright. All rights reserved.
    Journal of Applied Microbiology 08/2013; DOI:10.1111/jam.12332 · 2.39 Impact Factor
  • Source
    • "Freezing Containe r (Thermo Scientific, US) at a chamber temperature of À80 °C (cooling rate of À1 °C/min) without a nucleation step, and kept at À80 °C for 24 h. For each independent freeze-thaw assay, two ampoules of each condition were thawed at 25 °C and phage titer was determined by the soft agar overlay method in LB agar medium [8]. For lyophilization assays, frozen samples were dehydrated in a Heto FD4 model tray type freeze-dryer (LabEquipment, Denmark) at a condenser temperat ure of À50 °C for 48 h (pressure <1 Pa). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The aims of this study were to determine the stability of Podoviridae coliphage CA933P during lyophilization and storage in different media, and to establish similarities between the results obtained and those expected through mechanisms described for proteins stabilization during freeze-drying. PBS and SM buffer were assayed as lyophilization media. The effect of inorganic salts concentration as well as the addition of disaccharides on phage stability during freeze-drying and storage was also studied. The addition of low sucrose concentration (0.1 mol l(-1)) to SM buffer stabilized phage during freezing and drying steps of the lyophilization process, but higher sugar concentrations were detrimental to phage stability during freeze-drying. Sucrose stabilized phage during storage for at least 120 days. The lyoprotective effect of low concentrations of disaccharides during the drying step of the lyophilization of proteins as well as the stabilization of the freeze-dried product in time correlated with the results obtained for phage CA933P.
    Cryobiology 03/2013; 66(3). DOI:10.1016/j.cryobiol.2013.03.007 · 1.64 Impact Factor
Show more