Colonies of pure bacterial strains are highly dense cell structures that are organized in distinct and typical arrangements. The size, shape and variability of bacterial colonies are strongly dependent on the species and also influenced by environmental conditions. However, the spatial organization of individual cells is unknown for most strains. Furthermore, it is largely unclear how specific heterogeneous cell types at different locations in the colony contribute to the overall structure and how they may also influence the overall function of a colony. The study investigates the local diversification of bacterial colony structures by introducing a local biopsy technique. The biopsied cells were then analyzed by microbial flow cytometry and cytometric fingerprinting, which further diversified the biopsied samples into many heterogeneous cell states. This two-stage resolution insight into colony structure was performed on five bacterial strains: Bacillus subtilis, Paenibacillus polymyxa, Kocuria rhizophila, Stenotrophomonas rhizophila, and Pseudomonas citronellolis. The effects of biopsy tool size (27G needle and 10 μL, 200 μL, 1000 μL pipette tips) and sampling location on the precision of the technique were tested by using both gate setting along Gaussian distributions of subpopulations and a grid gating tool as well as the t-distributed Stochastic Neighbor Embedding (t-SNE) method. Cell cycle stages, the distribution of living and dead cells and different spore types and their abundancies were identified, revealing a considerable heterogeneity of the biopsied bacterial samples and at the same time the particular structure of a colony, as the cells in the biopsies at the different sites proved to be physiologically very different. The local separation of cell types by biopsy and the diversification of heterogeneous cell types by cytometric fingerprinting took only 15 to 45 minutes. Using the approach, further functions could be determined by downstream analysis through subsequent cell sorting. The study provides a high-resolution biopsy technique that explores the spatial distribution of cell types and their heterogeneous physiological cell states, allowing conclusions to be drawn from biopsy composition at different locations to overarching functions of the entire bacterial colony.
