Sterile membrane syringe filter

Two K. pneumoniae K23 strains (Kp-9068 and KPi4275) were used as hosts for bacteriophage isolation. Phages vB_KpnM_Seu621 and vB_KpnP_Dlv622 were isolated from the Chermyanka river water samples and phage KpS8 was isolated from sewage by a previously described method (Van Twest and Kropinski, 2009 ), with slight modifications. In brief, 15 ml of sewage or river water samples were centrifuged at 10,000 g for 15 min, and the supernatant was filtered using a 0.22-μm sterile membrane syringe filter (Millipore, United States). Filtered supernatant and 0.2 ml of early log-phase host cultures (OD_600nm = 0.3) were added to 15 ml of double-concentrated LB broth and incubated overnight with agitation (200 rpm) at 37°C to amplify the phages. Further, the culture was centrifuged at 10,000 g for 15 min and subsequently filtered through 0.22-μm filters. Obtained lysates were serially diluted in LB and spotted on double-layer agar plates of host-strains for phage detection and isolation. Three rounds of single plaque purification and re-infection of exponentially growing host strains yielded pure bacteriophage suspensions. Bacteriophage titers were determined using a double-agar overlay plaque assay (Mazzocco et al., 2009 ).