The overlay was prepared in a Falcon tube in conditions of sterility by mixing sterile LB-medium ( 20 mL ) containing agar ( 0.12 g ) at ca. 42 °C , with 20 mg/mL solution of X-gal ( 333 μL ) , 50 μg/mL solution of kanamycin ( 20 μL ) and 1 × 10 CFU/mL 8 of the S. typhimurium strain ( 100 μL ) that harbours reporter transcriptional lacZ-fusions to either the archetypal PhoP-activated gene virK ( 14028 virK : : lacZ ) or to a gene activated by the OmpR -- EnvZ two-compo-nent system , tppB ( 14028 tppB : : MudI ) .