2.7. Prophage detection in host bacteria after phage therapy
In order to evaluate the occurrence of lysogenic conversion (evaluate
if the tested phage has the ability to incorporate its own genome
in the bacterial genome), the mitomycin C test was applied, according
to Silva et al. (2014a), Silva et al. (2014b). The absence of a clear zone
after the exposure to mytomycin C was interpreted as an indicator of
the absence of prophages in the bacterial genomes. Three independent
assays were performed.
2.8. Determination of frequencies of spontaneous phage-bacterial mutants
Spontaneous A. salmonicida mutants resistant to bacteriophages were
isolated by plating on double-layer agar with the phage (109 PFU/mL per
plate), according to Filippov et al. (2011). To calculate the mutation frequencies,
mean numbers of mutants in 1 mL of culture (prepared from
the culture with phages) were divided by mean total numbers of live
bacterial cells (prepared from the culture without phages) (Filippov
et al., 2011).