The bacteriophage sensitive strains used during the production and quality control of BFC-1 are P. aeruginosa strain ‘573’, were isolated at the Eliava Institute of Bacteriophage, Microbiology and Virology (EIBMV) in the 1970s from bone marrow interstitial fluid, and S. aureus strain '13 S44 S′, isolated at the Brussels Burn Centre in 2006 from a burn wound. Initially, S. aureus strain Wood 60 (EIBMV collection) was used for propagation of phage ISP, but for the production of this cocktail the phage was propagated on S. aureus 13 S44 S. The absence of temperate phages from the host strains was tested as described in a separate section of this paper.
For bacteriophage isolation from natural samples such as sewage and river water, one millilitre of 10×concentrated LB Broth (Becton Dickinson), 1 ml ‘host bacteria’ suspension, containing 108 cfu in LB broth and 9 ml sewage or river water were mixed in a 14 ml sterile tube. This tube was incubated at 37°C for 1.5–2 h. Subsequently, 200 µl of chloroform (Sigma-Aldrich, Bornem, Belgium) was added and the tube was further incubated at 4°C for 1 h. The lysate was aspirated with a sterile 5 ml syringe and passed through a 0.45 µm membrane filter (Minisart, Sartorius, Vilvoorde, Belgium). Bacteriophages were titrated using the agar overlay method, as described above. All plaques with different morphology were touched with a sterile pipette tip, inoculated into 2 ml of sterile LB broth in 14 ml sterile tubes and incubated at 37°C for 2 h. Subsequently, 50 µl of chloroform was added and the tube(s) were incubated at 4°C for 1 h. For each tube, a dilution series (log(0)−log(−4)) was made in sterile 14 ml tubes filled with LB broth. Each dilution was titrated using the agar overlay method. Plates showing 1–10 plaques were analysed in detail. Again, all plaques with different morphology were touched with a sterile pipette tip, inoculated into 2 ml of sterile LB broth in 14 ml sterile tubes and incubated at 37°C for 2 h. This complete cycle was repeated until one plaque morphotype was obtained (homogeneous plaques).
In the case of bacteriophage ISP, which was isolated in the 1920s, porcelain, rather than membrane filters were employed.
For bacteriophage isolation from natural samples such as sewage and river water, one millilitre of 10×concentrated LB Broth (Becton Dickinson), 1 ml ‘host bacteria’ suspension, containing 108 cfu in LB broth and 9 ml sewage or river water were mixed in a 14 ml sterile tube. This tube was incubated at 37°C for 1.5–2 h. Subsequently, 200 µl of chloroform (Sigma-Aldrich, Bornem, Belgium) was added and the tube was further incubated at 4°C for 1 h. The lysate was aspirated with a sterile 5 ml syringe and passed through a 0.45 µm membrane filter (Minisart, Sartorius, Vilvoorde, Belgium). Bacteriophages were titrated using the agar overlay method, as described above. All plaques with different morphology were touched with a sterile pipette tip, inoculated into 2 ml of sterile LB broth in 14 ml sterile tubes and incubated at 37°C for 2 h. Subsequently, 50 µl of chloroform was added and the tube(s) were incubated at 4°C for 1 h. For each tube, a dilution series (log(0)−log(−4)) was made in sterile 14 ml tubes filled with LB broth. Each dilution was titrated using the agar overlay method. Plates showing 1–10 plaques were analysed in detail. Again, all plaques with different morphology were touched with a sterile pipette tip, inoculated into 2 ml of sterile LB broth in 14 ml sterile tubes and incubated at 37°C for 2 h. This complete cycle was repeated until one plaque morphotype was obtained (homogeneous plaques).
In the case of bacteriophage ISP, which was isolated in the 1920s, porcelain, rather than membrane filters were employed.
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