In a project conducted by ILVO (Belgium), a wide variety of bacterial species were recovered from the surface of a dairy pasteurizer after cleaning and disinfection (C&D). The biofilm-forming ability of these bacteria was determined in both single-species and various mixed-culture combinations. Some work related to this study has been published in Frontiers: "Synergistic interactions in multispecies biofilm combinations of bacterial isolates recovered from diverse food processing industries". Bacterial species were mixed in different combinations to assess the community biofilm mass and growth dynamics of individual species. ILVO and the University of Copenhagen conducted experiments aimed at revealing the structural characteristics and spatial organization of bacterial species within different mixed-species biofilms. In our research, we employed oligonucleotide FISH probes, each conjugated with a unique fluorescent dye: Cy5 for Stenotrophomonas rhizophila (B68), Cy3 for Bacillus licheniformis (B65), and FAM for Microbacterium lacticum (B30). C1 combination refers to a combination containing B68 and B30. Images of the biofilms formed on the coupons were captured using a confocal laser scanning microscope (LSM 800, Zeiss) with a Plan-Apochromat 63x/1.4 oil-immersion objective. Z-stacks were recorded to obtain three-dimensional (3D) images. Standard images were made with an image size of 1024 × 1024 pixels, corresponding to physical dimensions of 101.4 × 101.4 μm for each image. For each image, two separate channels were applied to detect any dual-species combination using a flexible detector (GaAsP-PMT) in the LSM 800 system. Representative 3D views of images were generated using the 3D model function in the ZEN system 3.7. Biofilms were grown in BHI for 24 h on plastic coupons. The samples were imaged at different time points: 6h, 12h, 18h and 24h. Each samples had three replicates and for each replicate imaging was performed from 3-6 different positions. Details of the oligonucleotide probes are given below: Name of the species Sequences Max. excitation Max. emission Fluorophores S. rhizophila B68 GGGCCTTTACCCCGCCA 649 nm 670 nm Cy5 B. licheniformis B65 ACCGCCTGCGCGCGCTT 550 nm 570 nm Cy3 M. lacticum B30 CCCCACCCTTTCGCTCC 495 nm 520 nm FAM