Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprinting
Abstract 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...
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Wiley
2025-05-01
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| Series: | Methods in Ecology and Evolution |
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| Online Access: | https://doi.org/10.1111/2041-210X.70022 |
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| author | Gorkhmaz Abbaszade Kathrin Stückrath Susann Müller |
| author_facet | Gorkhmaz Abbaszade Kathrin Stückrath Susann Müller |
| author_sort | Gorkhmaz Abbaszade |
| collection | DOAJ |
| description | Abstract 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. 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. 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. 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. |
| format | Article |
| id | doaj-art-08cefdfd0f6844d586269826d58a4abf |
| institution | OA Journals |
| issn | 2041-210X |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Wiley |
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| series | Methods in Ecology and Evolution |
| spelling | doaj-art-08cefdfd0f6844d586269826d58a4abf2025-08-20T02:16:06ZengWileyMethods in Ecology and Evolution2041-210X2025-05-0116597298710.1111/2041-210X.70022Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprintingGorkhmaz Abbaszade0Kathrin Stückrath1Susann Müller2Department of Applied Microbial Ecology Helmholtz‐Centre for Environmental Research—UFZ Leipzig GermanyDepartment of Applied Microbial Ecology Helmholtz‐Centre for Environmental Research—UFZ Leipzig GermanyDepartment of Applied Microbial Ecology Helmholtz‐Centre for Environmental Research—UFZ Leipzig GermanyAbstract 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. 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. 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. 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.https://doi.org/10.1111/2041-210X.70022cell heterogeneitycollective live formscolony biopsycytometric fingerprintingmicrobial flow cytometryphysiological cell states |
| spellingShingle | Gorkhmaz Abbaszade Kathrin Stückrath Susann Müller Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprinting Methods in Ecology and Evolution cell heterogeneity collective live forms colony biopsy cytometric fingerprinting microbial flow cytometry physiological cell states |
| title | Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprinting |
| title_full | Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprinting |
| title_fullStr | Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprinting |
| title_full_unstemmed | Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprinting |
| title_short | Bacterial colony biopsies: Spatial discrimination of heterogeneous cell types by cytometric fingerprinting |
| title_sort | bacterial colony biopsies spatial discrimination of heterogeneous cell types by cytometric fingerprinting |
| topic | cell heterogeneity collective live forms colony biopsy cytometric fingerprinting microbial flow cytometry physiological cell states |
| url | https://doi.org/10.1111/2041-210X.70022 |
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