The bacteriophages were isolated from sewage water sampled at the Käppala waste water treatment plant (location: WGS84: 59°21’22.2"N 18°13’45.3"E), recipient of waste water from Stockholm city including some hospitals, and kept at +4°C before processing. The sampling of water was approved by the owner, Käppalaförbundet, Box 3095, 181 03 Lidingö, Sweden, kappala@kappala.se (Phages are not considered to be protected species). The phages were amplified by mixing 50 ml of waste water with the same amount of double strength LB and 10 ml of a single ECOR strain bacteria cultured overnight. After incubation overnight at 30°C, 10 ml of the mixture was shaken with 1% v/v chloroform and left at room temperature for 30 minutes to kill the bacteria, centrifuged at 3000×g at +4°C for 15 minutes, and sterile filtered through a 0.45 μm membrane filter (Whatman, ref. no. 10462100). After checking the lysates for phages, the titre was measured in plaque assays. Sterile filtered phage lysates were diluted in SM buffer [30 ] or LB to five different dilutions (10-5–10-9). 100 μl of diluted phage and 200 μl of target bacteria were mixed with 2 ml SA, spread on pre-warmed LA plates, and incubated overnight at 30°C [31 (link)]. The harvested phages were selected according to their plaque morphology. Phages displaying large, clear and non-turbid plaques without a halo were classified as virulent. Phages were re-isolated by plaque purification from the LA plates when several phages on the same plate could be suspected. After additional plaque purifications, 25 virulent phages were saved and stored in 50% glycerol at -70°C as well as in LB at +4°C [32 (link)]. These phages were named according to guidelines in Kropinski et al. [33 (link)]. The six phages showing the broadest host range in the spot test (see below) were thus named as follows; vB_EcoP_SU10, vB_EcoP_SU16, vB_EcoP_SU27, vB_EcoP_SU32, vB_EcoP_SU57, and vB_EcoP_SU63, but only the last part of names is used in the following text, e.g. SU10, SU16 and so on.
Free full text: Click here