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.

    • "Virology (2015), Phage host range Host range analysis was performed as described elsewhere by double agar overlay assays and spot-on-the-lawn assays (Kropinski et al., 2009; Loessner and Busse, 1990). A total of 26 Listeria strains were tested, including six cell wall mutants (Supplementary Table S1). "
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    ABSTRACT: Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. Copyright © 2014 Elsevier Inc. All rights reserved.
    Virology 02/2015; 477. DOI:10.1016/j.virol.2014.12.035 · 3.28 Impact Factor
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    • "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. "
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    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). "
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    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; 115(6). DOI:10.1111/jam.12332 · 2.39 Impact Factor
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Aug 12, 2015