**12.1. Classical methods (isolation and selection of phage tolerant strains against the most aggressive phages from the dairy environment)**

In order to isolate phage-resistant mutants, a secondary culture method can be used [138], in which sensitive strains undergo selective pressure of their specific phages. Sensitive strains are inoculated in liquid medium and subsequently infected with suspensions of a selected lytic phage at specific titer. Liquid cultures exhibiting complete lysis are incubated for 24-48 hours (secondary growth). After incubation, bacteria are streaked on adequate solid medium. The grown colonies are consecutively cultured in liquid medium with the same selected phage during at least three rounds. Resultant isolates that are able to grow normally in the presence of the specific phage are considered as true phage-resistant mutants [139].

Another means of natural selection of phage-resistant strains was developed by Viscardi and colleagues [140]. The approach is based on flow-cytometry technique that senses and selects bacterial cells to which phage particles that have been added to the medium did not adsorb. Two detection methods have been designed, which rely on recognition of either specifically labeled anti-phage antibodies or fluorochrome-stained phages. The presented method is an attractive alternative to other means of isolating phage-resistant strains (described earlier). In the study, several different *Streptococcus thermophilus* strains were analyzed for their potential to develop spontaneous phage resistance that could be detected by flow-cytometry technique. The designed selection methods proved quite sensitive, as phage-resistant cells could be detected after only one selection round. Nonetheless, a tworound selection based on selection with anti-phage antibodies or labeled phages and then with unlabeled phage alone was more efficient in obtaining stable and proper phageresistant mutants. Phage adsorption assays determined that majority of the isolated mutants resisted phage infection at the level of phage adsorption. Moreover, several selection rounds using different labeled phages lead to isolating multi-phage resistant cells.

The great advantage of the method is its high sensitivity (detection of 2 out 107 cells) and high analysis rates (103 cells per second). As the occurrence of spontaneous phage-resistant cells is rather low in nature, the method allows increasing the level of detection of such mutants. Furthermore, the selected *S. thermophilus* mutants were resistant to phage attack throughout multiple generations, indicating the stability of this property. The novelty of the method is the short amount (several days) of time necessary for obtaining phage-resistant mutants. This creates a possibility of fast selection of new resistant starter strains in the presence of novel phages, which constantly break away from the current defense systems.
