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

Published online by Cambridge University Press:  04 February 2014

Christine A. Brantner ,
Ryan M. Hannah ,
James P. Burans  and
Robert K. Pope
Christine A. Brantner
Affiliation:
National Bioforensic Analysis Center, National Biodefense Analysis and Countermeasures Center, Fort Detrick, MD, 21702, USA
Ryan M. Hannah
Affiliation:
National Bioforensic Analysis Center, National Biodefense Analysis and Countermeasures Center, Fort Detrick, MD, 21702, USA
James P. Burans
Affiliation:
National Bioforensic Analysis Center, National Biodefense Analysis and Countermeasures Center, Fort Detrick, MD, 21702, USA
Robert K. Pope*
Affiliation:
National Bioforensic Analysis Center, National Biodefense Analysis and Countermeasures Center, Fort Detrick, MD, 21702, USA
*
*Corresponding author. E-mail: robert.pope@nbacc.dhs.gov
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Abstract

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.

Keywords

Bacillus anthracisBacillus cereusBacillus megateriumBacillus subtilisBacillus thuringiensisClostridium perfringenssporessterilizationultrastructureparaformaldehydeglutaraldehydeinactivationbiocontainment
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 1 , February 2014 , pp. 238 - 244
Copyright
Copyright © Microscopy Society of America 2014 

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