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Updated: Jan 21, 2026
06:39
Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry
Published on: June 30, 2023
1.6K
Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating
Marianna Karava1, Felix Bracharz1, Johannes Kabisch1
1Computer-aided Synthetic Biology, Institute for Biology, Technische Universität Darmstadt, Darmstadt, Germany.
Plos One
|July 30, 2019
Summary
Flow cytometry rapidly quantifies Bacillus subtilis endospore formation, revealing a double mutant
Area of Science:
- Microbiology
- Cell Biology
- Biotechnology
Background:
- Bacillus subtilis is a Gram-positive bacterium renowned for its ability to form endospores.
- Endospore formation in B. subtilis serves as a model for cellular differentiation and has biotechnological applications.
- Sporulating cultures comprise distinct subpopulations: vegetative cells, sporulating cells, and mature spores.
Purpose of the Study:
- To develop a rapid and accurate method for quantifying endospore formation in B. subtilis.
- To investigate the distribution of sporulating cell populations at a single-cell level.
- To monitor sporulation efficiency in genetically modified strains.
Main Methods:
- Utilized flow cytometry and fluorescence-activated cell sorting (FACS) with nucleic acid staining.
- Employed automated gating using Gaussian mixture modeling (GMM) for objective analysis.
- Applied the method to monitor sporulation in germination-deficient strains with genomic modifications.
Main Results:
- Observed decreased sporulation efficiency in strain Bs02018 (sfGFP surface display).
- Identified a double knockout mutant (spo0E and skfA, strain Bs02025) with significantly enhanced sporulation efficiency.
- Strain Bs02025 reached 80% spore content within 24 hours, compared to 18% for the control strain.
Conclusions:
- Flow cytometry combined with GMM provides a robust, high-throughput method for analyzing bacterial sporulation.
- Genetic modifications targeting spo0E and skfA can dramatically increase B. subtilis sporulation efficiency.
- The developed method facilitates the study of sporulation dynamics and strain optimization for biotechnological purposes.
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