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Biofilms were then stained with a live/dead stain (Bacterial viability kit, Life Technologies Inc., United States) for 30 min in the dark and cells were then visualized by CLSM (Olympus FV1200, Australia) with Ex 473 and 559 nm and Em 500 and 637 nm , for Syto-9 (green-live) and propidium iodide (red-dead) staining, respectively

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For the species-level analysis of Bifidobacterium , taxonomic assignments were conducted by aligning each 16S rRNA V3-V4 sequence to the RefSeq database of 16S rRNA gene sequences released by NCBI ( updated at 2023.4.15) using the qiime feature-classifier classify-consensus-blast command with the parameter --p-perc-identity 0.99