Trypticase soy agar with 5 sheep blood tsa 2

For the adhesion assessment each bacterial suspension (L. lactis or pathogen) was inoculated in triplicate to achieve a Ratio of Interaction (ROI; ratio of bacteria to cells) of 200:1. Microplates were incubated for 2 h at 37°C in 5% CO₂. After incubation, supernatants were discarded, and the non-adherent bacteria were removed by washing each well twice with PBS. Monolayers were lysed by freeze-thawing twice in 100 μL of sterilized water per well. To enumerate the adhesion bacteria cell lysates were serially diluted (1:10) with PBS and pour-plated on BHI agar for L. lactis, MacConkey agar (Oxoid, England) for E. coli, Staphylococci 110 agar (Biolife Italiana, Italy) for staphylococcal strains and Trypticase Soy Agar with 5% Sheep Blood (TSA II; BD, New Jersey) for streptococcal strains with the exception of S. agalactiae LMG 14838 that was plated on CHROMagar Orientation (BD, New Jersey) because of its non-hemolytic property. All plates were incubated at 37°C for up to 48 h to allow the development of visible colonies. The mean population values across three plates per well were calculated. The medians and interquartile ranges of the triplicate samples were then determined.