Bacillus anthracis spore suspensions: determination of stability and comparison of enumeration techniques

To determine the stability and variability in concentration of spore suspensions of Bacillus anthracis (BA) spore suspensions by comparing different methods of enumeration and to detect changes, if any, under different storage conditions. Plate and microscope counts were compared to measuring the ge...

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Bibliographic Details
Published in:Journal of applied microbiology 2008-05, Vol.104 (5), p.1442-1448
Main Authors: Almeida, J.L, Harper, B, Cole, K.D
Format: Article
Language:English
Subjects:
Anthrax - microbiology
Bacillus anthracis
Bacillus anthracis - genetics
Bacillus anthracis - physiology
Bacteriological Techniques
Biological and medical sciences
DNA extraction
DNA Primers - genetics
DNA, Bacterial - analysis
Fundamental and applied biological sciences. Psychology
Microbial Viability
Microbiology
Polymerase Chain Reaction - methods
Preservation, Biological
quantitative PCR
spores
Spores, Bacterial
stability
storage
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Summary:To determine the stability and variability in concentration of spore suspensions of Bacillus anthracis (BA) spore suspensions by comparing different methods of enumeration and to detect changes, if any, under different storage conditions. Plate and microscope counts were compared to measuring the genomic equivalents based on DNA content BA spore suspensions. We developed chemical methods to extract spore DNA and extra-spore (ES) DNA. DNA mass was determined by gel electrophoresis and QPCR assays were developed using the markers on the chromosome (rpoB) and the pXO1 plasmid (pag). The plate counts and microscope counts were very stable (for up to 900 days). The effect of freezing and the presence of additives in samples were tested for up to 300 days, and the results indicated that the additives tested and freezing did not decrease the viability or microscope counts. Bacillus anthracis spore suspensions can be stored for long periods of time without significant loss of viability or clumping. The content of ES DNA was variable and changed with time. The study shows that BA spore suspensions can be developed for reference materials providing a uniform basis for comparing detection equipment and results from different laboratories.
ISSN:1364-5072
1365-2672
DOI:10.1111/j.1365-2672.2007.03684.x