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Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry
Published on: June 30, 2023
Flow cytometry sorting protocol of Bacillus spore using ultraviolet laser and autofluorescence as main sorting
Christian Laflamme1, Daniel Verreault, Jim Ho
1Centre de recherche, Hôpital Laval, Institut Universitaire de cardiologie et de pneumologie de l'Université Laval, 2725 Chemin Ste-Foy, Québec, Québec, Canada.
Journal of Fluorescence
|October 13, 2006
Summary
Researchers developed a flow cytometry sorting protocol using ultraviolet (UV) autofluorescence to separate bacterial spores. This method effectively distinguishes between lower and higher autofluorescence levels in spore populations, demonstrating potential for viability assessment.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Microbiology
Background:
- The Ultraviolet (UV) Fluorescent Aerodynamic Particle Sizer (FLAPS) detects biological aerosol particles, like bacterial spores, via autofluorescence.
- Recent findings indicate a direct correlation between autofluorescence intensity and spore viability, with viable spores exhibiting higher UV autofluorescence.
- This study leverages flow cytometry to sort bacterial spores based on their UV-induced autofluorescence properties.
Purpose of the Study:
- To describe a flow cytometry sorting protocol for bacterial spores based on UV-induced autofluorescence.
- To investigate the separation of spore populations with distinct autofluorescence levels (lower level of autofluorescence (LLA) and higher level of autofluorescence (HLA)).
- To validate the sorting protocol using Bacillus subtilis var niger as a surrogate for Bacillus anthracis spores.
Main Methods:
- Utilized an EPICS ELITE ESP flow cytometer with a UV laser and cell sorter to replicate FLAPS optical properties.
- Employed autofluorescence intensity as the primary criterion for sorting Bacillus spores.
- Developed and described a laboratory protocol for sorting bacterial spores based on autofluorescence.
Main Results:
- Successfully sorted Bacillus spores into two distinct populations based on autofluorescence intensity.
- Achieved purity greater than 95% for each sorted spore population.
- Demonstrated the feasibility of separating spore populations using autofluorescence as the main sorting criterion.
Conclusions:
- The developed flow cytometry protocol effectively separates bacterial spores into distinct populations based on UV autofluorescence.
- Autofluorescence intensity serves as a reliable criterion for differentiating spore populations, potentially indicating viability differences.
- This method offers a promising approach for analyzing and sorting biological aerosol particles in real-time.
