Protocol for Isolation, Purification, Host-Range Determination, and Lytic Activity of Bacteriophages Against Pseudomonas aeruginosa v1

This protocol describes the laboratory procedures used for the isolation, purification, propagation, and characterization of bacteriophages targeting bacterial pathogens, with particular emphasis on Pseudomonas aeruginosa. The protocol includes phage enrichment from environmental and animal-derived samples, detection of phage activity using spot assays, quantitative phage enumeration using the double-layer agar plaque assay, and purification of individual phage isolates through successive single-plaque purification rounds. Purified phages are subsequently propagated and prepared as working lysates for downstream characterization and lytic activity assays. The protocol further describes the preliminary determination of phage host range against selected bacterial reference strains and the assessment of phage susceptibility among P. aeruginosa isolates. In addition, a 96-well microtiter plate crystal-violet assay is used to evaluate the ability of selected bacteriophages to disrupt preformed P. aeruginosa biofilms. Appropriate positive, negative, and process controls are incorporated throughout the procedures to ensure the reliability, reproducibility, and validity of the experimental results.