Inactivation and Ultrastructure Analysis of Bacillus spp. and Clostridium perfringens Spores

Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fi...

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Bibliographic Details
Published in:Microscopy and microanalysis 2014-02, Vol.20 (1), p.238-244
Main Authors: Brantner, Christine A., Hannah, Ryan M., Burans, James P., Pope, Robert K.
Format: Article
Language:English
Subjects:
Bacillus - drug effects
Bacillus - ultrastructure
Bacillus anthracis
Bacillus cereus
Bacillus megaterium
Bacillus subtilis
Bacillus thuringiensis
Biological Applications
Clostridium perfringens
Clostridium perfringens - drug effects
Clostridium perfringens - ultrastructure
Colony Count, Microbial
Environmental factors
Fixatives - pharmacology
Formaldehyde - pharmacology
Glutaral - pharmacology
Inactivation
Irradiation
Microbial Viability - drug effects
Microscopy, Electron, Scanning
Polymers - pharmacology
Scanning electron microscopy
Spores, Bacterial - drug effects
Spores, Bacterial - ultrastructure
Tuberculosis
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Summary:Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1–21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.
ISSN:1431-9276
1435-8115
DOI:10.1017/S1431927613013949