Application of direct plaque assay for detection and enumeration of bacteriophages of Bacteroides fragilis from contaminated-water samples

Córnax, Roberto; Moriñigo, Miguel A.; Paez, I G; Muñoz, M.; Borrego, Juan J.

doi:10.1128/aem.56.10.3170-3173.1990

Cited by 40 publications

(12 citation statements)

References 16 publications

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“…Methods for the detection and quantification of bacteriophages have been available ever since their discovery by Felix d’Herelle in 1917 [1]. These methods, based on the presence of lysis plaques in lawns of host bacteria growing in a double agar layer (DAL), were described in detail by Mark Adams in 1959 [2] and, with the addition of several modifications and improvements [3–7] they have constituted the workhorse of virus quantification until now.…”

Section: Introductionmentioning

confidence: 99%

Fast phage detection and quantification: An optical density-based approach

2019

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Since 1959 with the proposal of Double Agar Layer (DAL) method for phage detection and quantification, many sophisticated methods have emerged meanwhile. However, many of them are either too complex/expensive or insensitive to replace routine utilization of DAL method in clinical, environmental and industrial environments. For that purpose, we have explored an alternative method for the detection and quantification of bacteriophages that fulfills the criteria of being rapid, simple and inexpensive. In this paper we have developed a method based on the analysis of optical density kinetics in bacterial cultures exposed to phage-containing samples. Although the decrease in optical density caused by cell lysis was one of the first observable consequences of the effect of viral infection in bacterial cultures, the potential of the method for the assessment of phage abundance has never been fully exploited. In this work we carry out a detailed study of optical density kinetics in phage-infected bacterial cultures, as a function of both, phage abundance and initial concentration of the host organisms. In total, 90 different combinations of bacteria/phage concentrations have been used. The data obtained provide valuable information about sensitivity ranges, duration of the assay, percentages of inhibition and type of lysing behavior for each phage concentration. The method described can detect, as few as 10 phage particles per assay volume after a phage incubation period of 3.5h. The duration of the assay can be shortened to 45min at the expense of losing sensitivity and increasing the limit of detection to 10 8 pfu/ml. Despite using non-sophisticated technology, the method described has shown sensitivity and response time comparable to other high-end methods. The simplicity of the technology and of the analytical steps involved, make the system susceptible of miniaturization and automation for high-throughput applications which can be implemented in routine analysis in many environments.

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Section: Introductionmentioning

confidence: 99%

Fast phage detection and quantification: An optical density-based approach

2019

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“…Among the microbiological methods, the detection of bacteriophages infecting strain HSP40 of Bacteroides fragilis has very attractive features (7,12,15,21,(33)(34)(35). Strain HSP40 detects numbers of phages ranging from 10 1 to 10 2 per ml of urban sewage in some geographical areas, such as Southern Europe, South Africa, and Israel (3,4,9,13,32,35). However, it recovers lower numbers of phages in sewage from other geographical areas, such as the United States (22).…”

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confidence: 99%

Diversity ofBacteroides fragilisStrains in Their Capacity To Recover Phages from Human and Animal Wastes and from Fecally Polluted Wastewater

Puig

Queralt

Jofre

et al. 1999

Appl Environ Microbiol

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Great differences in capability to detect bacteriophages from urban sewage of the area of Barcelona existed among 115 strains ofBacteroides fragilis. The capability of six of the strains to detect phages in a variety of feces and wastewater was studied. Strains HSP40 and RYC4023 detected similar numbers of phages in urban sewage and did not detect phages in animal feces. The other four strains detected phages in the feces of different animal species and in wastewater of both human and animal origin. Strain RYC2056 recovered consistently higher counts than the other strains and also detected counts ranging from 101 to approximately 103phages per ml in urban sewage from different geographical areas. This strain detected bacteriophages in animal feces even though their relative concentration with respect to the other fecal indicators was significantly lower in wastewater polluted with animal feces than in urban sewage.

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“…Membrane filtration through microporous filters with pore diameters larger than viruses but smaller than bacteria is a simple method of removing unwanted bacteria (6,16). Tartera and Jofre (16) used membrane filters with 0.22-,um pores (Millipore Corp. Bedford, Mass.)…”

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confidence: 99%

“…Lipid solvents, such as ether and chloroform, can be used to inactivate bacteria but not viruses that do not contain lipid. Pretreating samples such as activated sludge, polluted river water, and sewage effluent with chloroform has produced large reductions in the numbers of indigenous bacteria and enhanced plaque resolution (6,8,16,17).…”

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confidence: 99%

Use of Hydrogen Peroxide Treatment and Crystal Violet Agar Plates for Selective Recovery of Bacteriophages from Natural Environments

Asghari

Farrah

Bitton

1992

Appl Environ Microbiol

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Hydrogen peroxide inactivated bacteriophages and bacteria at different rates. A concentration of 0.1% hydrogen peroxide reduced the numbers of several bacteria by an average of 94% but caused an average of 25% inactivation in the numbers of bacteriophages tested. Treating natural samples with hydrogen peroxide selectively reduced the indigenous bacterial flora and permitted better visualization of plaques of lawns of Escherichia coli C-3000. In some cases indigenous gram-positive bacteria were relatively resistant to hydrogen peroxide, but their growth could be limited by incorporation of crystal violet into the bottom agar used for plaque assays. The use of hydrogen peroxide treatment and crystal violet-containing plates permitted recovery of more phages from natural samples than did other procedures, such as chloroform pretreatment or the use of selective plating agar such as EC medium.

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Application of direct plaque assay for detection and enumeration of bacteriophages of Bacteroides fragilis from contaminated-water samples

Cited by 40 publications

References 16 publications

Fast phage detection and quantification: An optical density-based approach

Fast phage detection and quantification: An optical density-based approach

Diversity ofBacteroides fragilisStrains in Their Capacity To Recover Phages from Human and Animal Wastes and from Fecally Polluted Wastewater

Use of Hydrogen Peroxide Treatment and Crystal Violet Agar Plates for Selective Recovery of Bacteriophages from Natural Environments

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