Bacterial colony biopsies: spatial discrimination of heterogeneous cell types by cytometric fingerprinting
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. How specific heterogeneous cell types at different locations in a colony contribute to the overall structure and how they may influence the overall function of a colony is largely unknown. The study aims to investigate the local diversification of bacterial colony structures by introducing a local biopsy technique. The biopsied cells were analyzed by microbial flow cytometry and cytometric fingerprinting, which 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 {micro}L, 200 {micro}L, 1000 {micro}L pipette tips) and sampling location on the precision of the technique were tested by using both gates setting along Gaussian distributions of subpopulations and a grid gating tool as well as the t-distributed Stochastic Neighbor Embedding (t-SNE) method. The biopsy technique uncovered significant heterogeneity among the cells within bacterial colonies, identifying differences in cell cycle stages, the proportion of living and dead cells, and the abundance of spore types. Cells from different biopsy sites displayed distinct physiological states, revealing that colony structure is far more complex than previously understood. The techniques precision depends on the biopsy tool size, dye equilibration, and cell handling, underscoring the importance of method calibration. The biopsy method, combined with cytometric fingerprinting, provided insights within only 15 to 45 minutes and is universally applicable. 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. This method also facilitates downstream analysis through further cell sorting, offering a powerful approach for future functional investigations.