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


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.

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    • "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 · 3.32 Impact Factor
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    • "After enrichment the bacterial cells were first centrifuged at 5,000 ×g for 10 min, the supernatant was filtrated through 0.22 μm cellulose acetate filter paper. For plaque assay method, 1 mL filtered sample was mixed with 1 mL of host culture of each species and poured onto autoclaved petriplates after mixing the melted PYCa agar [29, 30]. Plates were then incubated at 30°C for 2 days as per the method of Petrovski et al. [13]. "
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    ABSTRACT: Activated sludge plants (ASP) are associated with the stable foaming problem worldwide. Apart from the physical and chemical treatment methods, biological treatment method has been least explored and may prove to be a novel and ecofriendly approach to tackle the problem of stable foam formation. In ASP Nocardia species are commonly found and are one of the major causes for forming sticky and stable foam. This study describes the isolation and characterization of three Nocardia bacteriophages NOC1, NOC2, and NOC3 for the control of Nocardia species. The bacteriophages isolated in this study have shown promising results in controlling foam producing bacterial growth under laboratory conditions, suggesting that it may prove useful in the field as an alternative biocontrol agent to reduce the foaming problem. To the best of our knowledge to date no work has been published from India related to biological approach for the control of foaming.
    07/2014; 2014:151952. DOI:10.1155/2014/151952
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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 · 9.30 Impact Factor
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