Palcam

The procedure was the same to the one described for L. monocytogenes pure culture biofilms, except that at day 1, only L. plantarum was inoculated (1 ml) into the wells with the coupons. After 24 h at 11°C, L. monocytogenes inoculum was added (1 ml) to the wells with L. plantarum in order to produce dual-species biofilms. This was needed since simultaneous co-inoculation resulted in complete absence of detectable cells of L. plantarum in the biofilms. The assays also included wells for pure culture L. plantarum biofilms in which, after 24 h, instead of L. monocytogenes inoculum, 1 ml of fresh TSB-YE was added. A similar procedure was performed when the biofilms were cultured in 1/10 diluted TSB-YE. Incubation proceeded at 11°C for 7 days without agitation.
After this period, biofilms were removed as described in the mono-species biofilm paragraph, except that suspensions from co-culture biofilms were additionally plated onto PALCAM (Biokar Diagnostics, Beauvais, France), in order to count L. monocytogenes CFU. Plates were incubated at 37°C for 48 h before counting the colonies. Lactobacillus plantarum was indirectly calculated by subtracting to the total CFU numbers in TSA-YE, the number of L. monocytogenes in PALCAM. Similarly to pure culture biofilms, two biological replicates with two technical replicates were always performed.