Permeabilization and hybridization protocols for rapid detection of Bacillus spores using fluorescence in situ hybridization

• Published in: Journal of microbiological methods Vol. 77; no. 1; pp. 29 - 36
• Main Authors: Filion, Geneviève, Laflamme, Christian, Turgeon, Nathalie, Ho, Jim, Duchaine, Caroline
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-04-2009; Elsevier
• Subjects: Bacillus; Bacillus - chemistry; Bacillus - cytology; Bacillus - isolation & purification; Bacillus - physiology; Bacillus spores; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Electron microscopy; Fluorescence in situ hybridization; Fluorescence microscopy; Fundamental and applied biological sciences. Psychology; In Situ Hybridization, Fluorescence - methods; Microbiology; Permeability; Permeabilization; Spores, Bacterial - chemistry; Spores, Bacterial - cytology; Spores, Bacterial - isolation & purification; Bacillus spores; Permeabilization; Electron microscopy; Fluorescence microscopy; Fluorescence in situ hybridization; Bacillales; Bacillus; Bacteria; Bacillaceae; Detection; Spores; Microbiological method
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2008.12.009

Background: Fluorescence
in situ hybridization (FISH) is not adapted for the detection of bacterial spores because of their resistance to conventional permeabilization treatments. Since spore-forming bacteria have important ecological, economical, and medical roles, their
in situ detection needs to be improved. The aim of this study was to develop rapid and effective protocols to permeabilize
Bacillus spores in order to apply the FISH technique.
Methods: Permeabilization protocols were developed for three species of
Bacillus spores. Hybridization was performed using universal and specific probes. Surface structural analysis of the permeabilization treatments was performed using scanning electron microscopy.
Results: With the proposed protocols,
Bacillus spores can be labeled in less than 1 h. The scanning microscopy showed some visible structural differences between the permeabilized spores compared to intact ones.
Conclusion: For the first time, rapid and effective protocols to detect
Bacillus spores by FISH were developed and can be applied to study
Bacillus spores using
in situ labeling within 1 h. Previously published
in situ hybridization protocols have never reached or been close to the currently described rapidity. This work will contribute to the possibility of near real time detection of biological threats that may be present as spores.