Fluorometric detection of phages in liquid media: Application to turbid samples.

During the last years there has been a growing interest in the development of methods for phage detection and quantification in environmental, public health and industrial sectors. Good methods of phage monitoring contribute to progress in phage therapies, biocontrol and food safety studies. They have also been used to indicate the possible presence of microbiological hazards in drinking and recreational waters, and are an essential tool to prevent failure of microbe-based industrial bioreactors. Many of the sophisticated methods that have emerged to cover these needs are strongly hampered by the presence of turbidity in the samples, that results in decreased sensitivity. To avoid this, time consuming pretreatment steps must often be included that increase the overall complexity of the assays and the time required to perform them. With this in mind, we have explored an alternative method that fulfills the criteria of being simple, rapid and inexpensive and can be used to perform analysis in turbid media without any pretreatment steps. In this paper we develop a method that monitors lysis of an indicator culture when exposed to samples containing the target phage. The method is based on the properties of resazurin, a redox dye that becomes fluorescent when reduced by an active microbial culture. We analyzed the fluorescence kinetics of non-turbid phage-infected bacterial cultures as a function of both, phage abundance and initial cell concentration. For this purpose, different phage/host combinations were used and then, the addition of resazurin at different times (0, 30 and 60 min) was carefully evaluated for each phage/host combination, thus providing data for 168 combinations in total. Next, selected phage/host combinations were tested over 4 different turbidity models: 0, 1000, 2000 Nephelometric Turbidity Units (NTU) as well as in milk. The data obtained provided information about the duration of the assay and sensitivity thresholds in matrices with different turbidity grades. The results obtained indicate that the method can detect as few as 10 phage particles per assay volume within 3.5 h. If sensitivity is not an issue and the threshold of detection is increased to 107 phages the assay is considerably shortened, providing reliable results in only 40 min. Overall, the detection approach proposed in this work provides a simple, rapid and inexpensive solution that compares favorably, in terms of performance, with other high-end methods.

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

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.