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. 2007 Sep;70(3):442-51.
doi: 10.1016/j.mimet.2007.05.019. Epub 2007 Jun 14.

Supercritical carbon dioxide and hydrogen peroxide cause mild changes in spore structures associated with high killing rate of Bacillus anthracis

Jian Zhang  1 , Nishita Dalal, Michael A Matthews, Lashanda N Waller, Clint Saunders, Karen F Fox, Alvin Fox
Affiliations

Supercritical carbon dioxide and hydrogen peroxide cause mild changes in spore structures associated with high killing rate of Bacillus anthracis

Jian Zhang et al. J Microbiol Methods. 2007 Sep.

Abstract

The present work examines chemical and structural response in B. anthracis spores killed by a mixture of supercritical carbon dioxide (SCCO(2)) and hydrogen peroxide (H(2)O(2)). Deactivation of 6-log of B. anthracis spores by SCCO(2)+H(2)O(2) was demonstrated, but changes in structure were observed in only a small portion of spores. Results from phase contrast microscopy proved that this treatment is mild and does not trigger germination-like changes. TEM imaging revealed mild damage in a portion of spores while the majority remained intact. Dipicolinic acid (DPA) analysis showed that <10% of the DPA was released from the spore core into the external milieu, further demonstrating only modest damage to the spores. Confocal fluorescent microscopy, assessing uptake of DNA-binding dyes, directly demonstrated compromise of the permeability barrier. However, the magnitude of uptake was small compared to spores that had been autoclaved. This work suggests that SCCO(2)+H(2)O(2) is quite mild compared to other sterilization methods, which has major implications in its application. These results provide some insight on the possible interactions between spores and the SCCO(2)+H(2)O(2) sterilization process.

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Figures

Figure 1
Figure 1
Schematic of supercritical CO2 apparatus
Figure 2
Figure 2
Phase contrast images of untreated, pure CO2 processed, and CO2+H2O2 processed spores
Figure 3
Figure 3
TEM images of B. anthracis spores
Figure 4
Figure 4
DPA release from B. anthracis spores. (a) Controls and spores treated with CO2+H2O2. (b) Controls and spores treated with pure CO2.
Figure 5
Figure 5
Fluorescent images of BacLight stained B. anthracis spores
Figure 5
Figure 5
Fluorescent images of BacLight stained B. anthracis spores
Figure 6
Figure 6
Percentages of different staining patterns of B. anthracis spores
See this image and copyright information in PMC

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