Y-005. A Method for Growing Bacillus anthracis in a Spore-Free Culture for Use in Molecular Applications Outside of the Bio Safety Level 3 Environment

D. S. King1, V. A. Luna1, A. Cannons1, P. Amuso2, J. Cattani1;
1Ctr. for Biological Defense, Univ. of South Florida, Tampa, FL, 2Florida Dept of Hlth., Tampa, FL.

In response to adverse environmental conditions, Bacillus anthracis produces endospores whose presence often precludes molecular bio-forensic analysis of isolates because analytical instruments typically are not located within the BSL-3 laboratory. We wanted to develop a method for producing a spore-deficient culture that could be lysed and safely removed from the BSL-3 to a BSL-2 environment. Several B. anthracis strains were grown on blood agar (BA) 24 hours at 37°C under anaerobic, microaerophilic and enriched CO2 atmospheres. Growth from a colony edge was used to inoculate heart infusion broth supplemented with horse serum and sodium bicarbonate (SHIB) and tryptic soy broth (TSB). Growth and spore production after 4 to 6 hours incubation were compared. Microscopic slides were prepared, stained with malachite green and examined. Cells were either embedded in low-melting point agarose and processed for PFGE, or lysed. Plugs or lysed cells were then heat-shocked (80°C) and cultured on BA. Plates were examined for growth daily for up to 7 days. Optimal conditions for spore-deficient cultures were 37°C, microaerophilic atmosphere, and 4 hour incubation in broth. In both broths, three strains produced sufficient cells for molecular work. One strain grew slowly in SHIB. None of the lysed cell preparations or plugs yielded live B. anthracis for any of the strains yet all were acceptable for Riboprint® and PFGE typing. This novel scheme for growing spore-free ,B. anthracis can be used to analyze DNA samples of B. anthracis safely outside of the BSL-3 environment and prevent instrument contamination.