Abstract
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 108 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.
Highlights
Methods for the detection and quantification of bacteriophages have been available ever since their discovery by Felix d’Herelle in 1917 [1]
The decrease in optical density caused by cell lysis was one of the first observable consequences of the effect of viral infection of bacterial cultures, the potential of the method for the assessment of phage abundance has never been fully exploited
In order to check the suitability of optical density measurements for the detection of low phage concentrations we carried out a series of experiments in which different concentrations of bacteria (105, 5x105, 106, 5x106, 107, 2.5x107, 5x107, 108, 2.5x108 and 5x108 colony forming unit per mL (cfu/mL)) were exposed to different concentrations of phage (0, 5x101, 5x102, 5x103, 5x104, 5x105, 5x106, 5x107, and 5x108 pfu/mL)
Summary
Methods for the detection and quantification of bacteriophages have been available ever since their discovery by Felix d’Herelle in 1917 [1]. Phage detection using optical density kinetics and analysis, decision to publish, or preparation of the manuscript
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