Purification of bacteriophages by dilution streaking double agar layer (DS-DAL) assay

We describe a simple modification of the classical double-agar-layer (DAL) assay that integrates Robert Koch's dilution-streaking to improve spatial separation of individual plaques and streamline the isolation of purified phages. In the method, named DS-DAL (dilution-streaking-DAL), a clarified phage lysate isolated against its specific host is first dilution-streaked across the surface of hard agar plates (1.5%); then the corresponding host-containing soft agar (0.75%) overlay is poured over the air-dried streaked surface, and the plates are incubated until plaques form along the streak tracks. We applied the technique to purify host-specific coliphages and Shigella phages intended for phage therapy in poultry, as well as polyvalent phages against Aeromonas for aquaculture applications. The approach produced discrete, well-spaced plaques with undiluted samples in the second or third line of the dilution streak (depending on the initial titer values), requiring only one plate to purify the phage, compared with 7-8 plates required for that many dilutions in conventional overlay plating. Thus, the dilution-streak DAL is a low-cost assay for rapid phage purification that requires no specialized equipment and is compatible with routine downstream workflows in both research and applied settings.