Procurement of spore-free Bacillus anthracis for molecular typing outside of BSL3 environment.

Aims: To (i) develop a protocol that would eliminate or greatly reduce sporulation within Bacillus anthracis vegetative cells, and (ii) harvest an adequate number of cells and sufficient DNA suitable for molecular methods including Riboprint (R) analysis and pulse field gel electrophoresis (PFGE). Methods and Results: Seven strains of B. anthracis (Ames, French B2, Heluky, Kruger, Pasteur, Sterne, and Vollum) were grown at 37, 42 and 45 degrees C under normal air, enhanced CO(2), microaerophilic, and anaerobic conditions on solid media and subcultured in two broths with and without supplements. The bacterial cells were centrifuged and washed. Slides made from the cell pellets were stained with Malachite Green and observed for the presence of spores. Cell preparations were subjected to 80 degrees C for 30 min and processed for and analysed by either Riboprinter (R) or PFGE. Multiple pellets of each strain were processed, stained, placed onto solid culture media, incubated for 7 days and observed for growth. The cell preparations yielded clear and reproducible results with both molecular methods. None of the cell preparations yielded growth on the culture media. Conclusions: This method eliminated viable spores in cell preparations of B. anthracis, yet still allowed the growth of vegetative cells to provide sufficient DNA suitable for analysis by Riboprinter (R) and PFGE. Significance and Impact of the Study: This method will provide safe cell preparations, prevent instrument contamination, and may be useful for other aerobic and anaerobic spore-formers.