Publication Details
Category Text Publication
Reference Category Journals
DOI 10.1111/2041-210x.70022
Licence creative commons licence
Title (Primary) Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprinting
Author Abbaszade, G.; Stückrath, K.; Müller, S.
Source Titel Methods in Ecology and Evolution
Year 2025
Department AME
Volume 16
Issue 5
Page From 972
Page To 987
Language englisch
Topic T7 Bioeconomy
Data and Software links https://doi.org/10.5281/zenodo.13791365
Supplements https://besjournals.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.1111%2F2041-210X.70022&file=mee370022-sup-0001-Supinfo.pdf
Keywords cell heterogeneity; collective live forms; colony biopsy; cytometric fingerprinting; microbial flow cytometry; physiological cell states
Abstract
  1. Colonies of pure bacterial strains are highly dense cellular structures organised in distinct and typical arrangements. The size, shape and variability of bacterial colonies are strongly species-dependent and also influenced by environmental conditions. However, the spatial organization of individual cells is unknown for most strains. By introducing a local biopsy technique, this study aimed to provide a means to study the local diversification of the structure of bacterial colonies.
  2. Cells were biopsied at different sites of a colony and analysed by microbial flow cytometry using cytometric fingerprints for both fixed and viable cells, which divided the biopsied samples into many heterogeneous cell states. This two-step resolution 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, 200, and 1000 μL pipette tips) and sampling site on the precision of the technique were tested by using both gate settings along Gaussian distributions of subpopulations and a grid gating tool, as well as the t-distributed stochastic neighbour embedding (t-SNE) method.
  3. The biopsy technique uncovered significant heterogeneity among the cells within bacterial colonies, identifying differences in cell cycle stages, the proportion of live 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 technique's 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 15–45 min and is universally applicable.
  4. 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 sites 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.
Persistent UFZ Identifier https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=30619
Abbaszade, G., Stückrath, K., Müller, S. (2025):
Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprinting
Methods Ecol. Evol. 16 (5), 972 - 987 10.1111/2041-210x.70022