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.
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    • "After incubation, the culture was added NaCl 5% for 30 min at 4°C, centrifuged twice at 4,000 ×g for 15 min at 4°C, the supernatant was collected into a sterile flask and filtered through a sterile 0.45-µm membrane filter (Fisher Scientific). To detect the Hoa et al. 3605 presence of phage in the filtrate, spot testing was performed as described previously by Kropinski et al. (2009). Phage preparations were obtained and stored at 4°C as described by Jamalludeen et al. (2009b). "

    Preview · Article · Oct 2014 · African journal of microbiology research
    • "Many of the methods described to isolate spontaneous phage-resistant mutants are derived from the classical plaque assay with double-layer agar (DLA) (Adams, 1959; Gratia, 1936). This method basically consists in mixing appropriate dilutions of both bacteria and phage in molten agar or agarose (the top overlay), which in turn, is evenly distributed on top of a solidified standard medium (the bottom underlay) (Kropinski et al., 2009). Using the standard DLA-system, in combination with high titers of a lytic phage that will kill almost all the bacterial population, it is possible to recover single colonies that further tests will confirm if they are phage-resistant. "
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    ABSTRACT: A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidate-resistant mutants.
    No preview · Article · Aug 2014 · Journal of Microbiological Methods
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