Enumeration of Bacteriophages Using the Small Drop Plaque Assay System

Mazzocco, Amanda; Waddell, Thomas E.; Lingohr, Erika J.; Johnson, Roger P.

doi:10.1007/978-1-60327-164-6_9

Cited by 168 publications

(135 citation statements)

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“…P. larvae strains were cultivated on Columbia sheep blood agar (BD) at 37°C with 5% CO 2 . For liquid culture, P. larvae strains were grown in brain heart infusion (BHI) medium (37 g (12).…”

Section: Methodsmentioning

confidence: 99%

“…For phage isolation, the cultures were mixed with 5% (vol/vol) CHCl 3 and shaken for 15 min at 37°C. The aqueous phase was centrifuged at 12,000 ϫ g for 15 min, and the supernatant was used for enumeration of bacteriophages in titration assays with the small-drop plaque assay system (12). The specific burst size of bacteriophage HB10c2 was calculated with the equation B HB10c2 ϭ log(PFU 0 /PFU)/ log(G P. larvae ), where the burst size (B) was calculated by the PFU at time zero (PFU 0 ) and the PFU after incubation (PFU) with regard to the number of host generations (G).…”

mentioning

confidence: 99%

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Paenibacilluslarvae-Directed Bacteriophage HB10c2 and Its Application in American Foulbrood-Affected Honey Bee Larvae

Beims

Wittmann

Bunk

et al. 2015

Appl Environ Microbiol

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dPaenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious honey bee brood bacterial disease. We isolated and characterized P. larvae-directed bacteriophages and developed criteria for safe phage therapy. Whole-genome analysis of a highly lytic virus of the family Siphoviridae (HB10c2) provided a detailed safety profile and uncovered its lysogenic nature and a putative beta-lactamase-like protein. To rate its antagonistic activity against the pathogens targeted and to specify potentially harmful effects on the bee population and the environment, P. larvae genotypes ERIC I to IV, representatives of the bee gut microbiota, and a broad panel of members of the order Bacillales were analyzed for phage HB10c2-induced lysis. Breeding assays with infected bee larvae revealed that the in vitro phage activity observed was not predictive of the real-life scenario and therapeutic efficacy. On the basis of the disclosed P. larvae-bacteriophage coevolution, we discuss the future prospects of AFB phage therapy.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Paenibacilluslarvae-Directed Bacteriophage HB10c2 and Its Application in American Foulbrood-Affected Honey Bee Larvae

Beims

Wittmann

Bunk

et al. 2015

Appl Environ Microbiol

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show abstract

“…After an overnight incubation, the overlay agar was removed from the plates and stored at 4°C overnight before being centrifuged at 12,000 g. The supernatant was filter sterilized through a 0.22 lm filter, and phages were enumerated using the small drop plaque assay system. (32) This supernatant became the crude phage lysate which was used in future experiments.…”

Section: Bacteriophage Isolation Propagation and Storagementioning

confidence: 99%

Bacteriophage Delivery by Nebulization and Efficacy Against Phenotypically DiversePseudomonas aeruginosafrom Cystic Fibrosis Patients

Sahota

Smith

Radhakrishnan

et al. 2015

Journal of Aerosol Medicine and Pulmonary Drug Delivery

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“…This method allows description of a spotting bactericidal host range (56) and relies on the formation of visualized plaques and/or lysis zones encompassing the phage suspension spot, allowing analysis of numerous samples (57,58). Among the tested strains, 46 were infected by at least one tectivirus (Table 5).…”

Section: Figmentioning

confidence: 99%

Prevalence, Genetic Diversity, and Host Range of Tectiviruses among Members of the Bacillus cereus Group

Gillis

Mahillon

2014

Appl Environ Microbiol

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and Wip1 are tectiviruses preying on the Bacillus cereus group. Despite the significant contributions of phages in different biological processes, little is known about the dealings taking place between tectiviruses and their Gram-positive bacterial hosts. Therefore, this work focuses on characterizing the interactions between tectiviruses and the B. cereus group by assessing their occurrence and genetic diversity and evaluating their host range. To study the occurrence of tectiviruses in the B. cereus group, 2,000 isolates were evaluated using primers designed to be specific to two variable regions detected in previously described elements. PCR and propagation tests revealed that tectivirus-like elements occurred in less than 3% of the isolates. Regardless of this limited distribution, several novel tectiviruses were found, and partial DNA sequencing indicated that a greater diversity exists within the family Tectiviridae. Analyses of the selected variable regions, along with their host range, showed that tectiviruses in the B. cereus group can be clustered mainly into two different groups: the ones infecting B. anthracis and those isolated from other B. cereus group members. In order to address the host range of some novel tectiviruses, 120 strains were tested for sensitivity. The results showed that all the tested tectiviruses produced lysis in at least one B. cereus sensu lato strain. Moreover, no simple relationship between the infection patterns of the tectiviruses and their diversity was found.

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Enumeration of Bacteriophages Using the Small Drop Plaque Assay System

Cited by 168 publications

References 1 publication

Paenibacilluslarvae-Directed Bacteriophage HB10c2 and Its Application in American Foulbrood-Affected Honey Bee Larvae

Paenibacilluslarvae-Directed Bacteriophage HB10c2 and Its Application in American Foulbrood-Affected Honey Bee Larvae

Bacteriophage Delivery by Nebulization and Efficacy Against Phenotypically DiversePseudomonas aeruginosafrom Cystic Fibrosis Patients

Prevalence, Genetic Diversity, and Host Range of Tectiviruses among Members of the Bacillus cereus Group

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