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Flow Cytometry Sorting Protocol of Bacillus Spore Using Ultraviolet Laser and Autofluorescence as Main Sorting Criterion

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Abstract

The ultraviolet (UV) Fluorescent Aerodynamic Particle Sizer (FLAPS), a flow cytometer-like apparatus was developed by the Canadian Department of National Defence for real-time detection of autofluorescence of biological aerosol particles such as bacterial spores. The direct relation between autofluorescence intensity and viability has recently been reported and viable spore are more autofluorescent in UV (Laflamme, Frontiers in Bioscience). The goal of this manuscript is to describe a flow cytometry sorting protocol based on UV-induced autofluorescence. An EPICS® ELITE ESP flow cytometer equipped with a UV laser and cell sorter was used to mimic the optical properties of FLAPS and to study the two extremes of a spore population according to its autofluorescence (lower level of autofluorescence (LLA) and higher level of autofluorescence (HLA) spores). Bacillus subtilis var niger was used as a surrogate for Bacillus anthracis spores and sorted using autofluorescence intensity as the main criterion. The protocol developed in our laboratory to sort Bacillus spores according to their autofluorescence properties is described. Purity of each sorted population was greater than 95%. Using autofluorescence as the main criterion, we demonstrate that it is possible to separate two distinct spore populations.

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Acknowledgements

These studies were undertaken under a Department of National Defence (DND) contract W7702-00R802/001/EDM: Dr. J. Ho was the scientific authority acting for DND. Initial ideas for these studies originated from previous work performed at DND. The authors are thankful to Dr. André Darveau and Maurice Dufour for their insightful review of the manuscript. Christian Laflamme received a Natural Sciences and Engineering Research Council of Canada (NSERC)/Institut de recherche Robert-Sauvé en santé et sécurité du travail (IRSST) studentship. Dr. C. Duchaine acknowledges an Institut de Recherche Robert-Sauvé en Santé et Sécurité du Travail/Canadian Institutes of Health Research Scholarship.

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Authors and Affiliations

  1. Centre de recherche, Hôpital Laval, Institut Universitaire de cardiologie et de pneumologie de l’Université Laval, 2725 Chemin Ste-Foy, Québec, Québec, Canada

    Christian Laflamme, Daniel Verreault & Caroline Duchaine

  2. Biological detection group, Defence R&D Canada Suffield, CFB Suffield, Ralston, G1V 4G5, Alberta, Canada

    Jim Ho

  3. Département de biochimie et de microbiologie, Université Laval, Québec, Québec, Canada

    Caroline Duchaine

Authors
  1. Christian Laflamme
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  2. Daniel Verreault
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  3. Jim Ho
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  4. Caroline Duchaine
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Correspondence to Caroline Duchaine.

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Laflamme, C., Verreault, D., Ho, J. et al. Flow Cytometry Sorting Protocol of Bacillus Spore Using Ultraviolet Laser and Autofluorescence as Main Sorting Criterion. J Fluoresc 16, 733–737 (2006). https://doi.org/10.1007/s10895-006-0129-1

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  • DOI: https://doi.org/10.1007/s10895-006-0129-1

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