Immunology and Infectious Diseases

Denis Rajnovic is a third year PhD student enrolled at the Autonomous University of Barcelona (UAB), Spain. Here at UAB, he is a part of the microbial biosensor group in which they tend to develop sensitive, cheap and fast detection tools for industrial or public health applications. His work is to develop microbial detection and quantification methods in clear and turbid media or on paper, by using optical, colorimetric or fluorescence readings. Also, he is part of a diagnostic group at the University Hospital of Vall d'Hebron in Barcelona, Spain. Here, the focus is to develop diagnostic and monitoring tools for infectious microorganisms. His role is to implement and optimize different colorimetric or fluorescent immunological assays for virus detection and to make it as sensitive and fast as possible. During the academic year, he hold practical classes in basic microbiology for the UAB students.

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.