Determination of phage host range

To assess the lytic spectrum of the accumulated bacteriophage, the double-layer method was used. As a test culture, 14 strains of Klebsiella pneumoniae were used. A suspension of Klebsiella pneumoniae cells in physiological salt solution was prepared. The concentration of cells in suspension is approximately 10⁹ cfu/ml. 0.2 ml of culture were added to tubes with soft agar (0.6%), mixed, poured on Petri dish with agar and allowed to solidify. Then, the phage suspension in several dilutions was spotted on the soft agar. Petri dishes were incubated for 24 h at + 37° C in a thermostat (Binder GmbH, Germany). After incubation, the presence of lysis spots was assessed. To verify that the formation of lysis spots was caused by the lytic action of the phage, bacteriophage plaque assays were performed as described above. Moreover, an analysis of lysis spots on a JEM-1011 electron microscope (JEOL, Japan) was performed. For this, a fragment of the agar plate was taken from the lysis spot and placed in a 0.1 ml drop of physiological saline. Incubated for 25–30 min. After that, the agar plate was removed and electron microscopy of the droplet was carried out. The presence of bacteriophages in the test material was evaluated.