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This invention relates to the field of biofilm microbial imaging and lineage analysis technology, and discloses a microbial analysis and processing method. The three-dimensional lineage tracking of individual microorganisms includes: identifying individual microorganisms in the biofilm through three-dimensional connectivity analysis; establishing individual movement trajectories across time points using a three-dimensional Kalman filter algorithm; detecting division events and establishing parent-child lineage associations based on trajectory changes, volume conservation, and spatial proximity; and finally assembling a four-dimensional spatiotemporal lineage diagram structure to represent the complete microbial proliferation and evolution process. This invention solves the technical problems of broken three-dimensional biofilm lineage tracking and loss of spatial diffusion trajectories in traditional two-dimensional analysis methods.