2.2. Phage Isolation, Propagation and Purification Techniques

Phage isolation was performed by using the enrichment of phages in the source material technique as described previously [13], using a local isolate, Pantoea agglomerans strain AUR, as a host. Phage titration was performed by using the soft agar overlay method described by Adams [29], with minor modifications. Briefly, 0.1 mL of diluted phage suspension was mixed with 0.5 mL of indicator cells (OD₆₀₀–0.5). The mixture then was added to 2.5 mL of 0.4% (w/v) soft agar and poured over the 1.2% LB agar plate as a uniform layer. The plates were incubated 1–10 days at 3–40°C before the enumeration and measurement of plaques. For comparison of the phage and bacterial counts in the different zones (lysis, halo and bacterial zone), an equally large surface of the top agar was removed from each zone, suspended in 0.1 mL of LB-medium and vortexed. Plaque forming units were determined using the double agar method. Initial AAS23 purification was performed by five consecutive transfers of phage from individual plaques to new bacterial cell lawns. Notably, as AAS23 propagated poorly in a liquid broth, the propagation of phage was performed by the soft agar overlay method, as described previously [30], using Pantoea agglomerans strain AUR as a host. The phage purification was performed using a CsCl step gradient [31], as described by Šimoliūnas et al. [28]. The adsorption tests were carried out as described by Kropinski [32]. For determination of the efficiency of plating (EOP), high-titer phage stocks were diluted and plated in triplicate. Plates incubated at 3–40 °C were read after 1–10 days of incubation. The temperature at which the largest number of plaques formed was taken as the standard for the EOP calculation.