The Sensitivity of Pseudomonas Agar Plaque Assay in the Isolation of Bacteriophage ?6 in the Environment: A pilot study

Background: Bacteriophage Φ6 is a lipid-enveloped dsRNA bacteriophage. The limitations in our knowledge of how this bacteriophage occurs in the environment are limited by non-selective isolation techniques. Research on finding phages in the environment in the past has employed the Double Agar Layer (DAL) plaque assay using Tryptic Soy Agar (TSA), a non-selective media. The bacterial host for bacteriophage Φ6 is Pseudomonas syringae. In this study, we tested Pseudomonas Agar, a selective media that suppresses the growth of bacteria except Pseudomonas species, in the standard double agar layer plaque assay for Φ6. Methods: DAL plaque assays were performed to determine the sensitivity of both Tryptic Soy Agar (TSA) and Pseudomonas Agar (PA) for determining the titer of pure bacteriophage Φ6 stocks. We used Pseudomonas syringae (HB10Y) as the host, and the plaque formation on both agars was compared. Following the evaluation of PA with pure Φ6 stocks, PA effectiveness for Φ6 isolation from environmental samples was tested in spiked waters obtained from irrigation ponds at an agricultural farm. Results: Comparison of TSA and PA using pure Φ6 cultured in the laboratory and spiked environmental samples showed that PA agar can detect bacteriophage Φ6 as well as the standard DAL assay using TSA. On PA, formation of clear visible plaques comparable to the plaques formed using TSA was observed. Conclusions: Pseudomonas Agar can be used for the isolation of bacteriophage Φ6 in environmental samples. This may enhance the detection of these phages in the environment