Bacteriophages specifically targeting different strains of bacteria can be isolated from urban sewage using properly modified enrichment techniques. This chapter provides a detailed protocol for isolation of Proteus mirabilis-specific bacteriophages. Briefly, prefiltered sewage is mixed with double-concentrated tryptic soy broth containing the target strain and incubated. Subsequently, the suspension is spread on phage nutrient agar, and if needed, supplemented with swarming motility inhibitor, for the induction of bacterial growth and phage multiplication. Phages infecting bacteria are identified by plaques (patches of dead bacteria) in the confluent bacterial lawn. A pure phage preparation is obtained by cutting out a single plaque from a double-layer agar plate and subsequent virus propagation five times on a given P. mirabilis strain.
Keywords: Bacteriophage; Bacteriophage isolation; Double-layer agar method; Enrichment technique; Plaque; Proteus mirabilis; Sewage.