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a ’ t FD-302a (Rev. 10-6-95) 279A-WF-222936-302 Continuation of FD-302 of ae ,On 05/12/2006 _ , Page 3 opined if some one adapted toxin fermentation protocol in order to produce Bacillus anthracis spores, it would probably take 2-3 days in order to produce spores; however, the spore yield would be low. As it related to the toxin studies at USAMRIID, after fermentation a small _ quantity of its contents was viewed microscopically. [-~ Jadvisea almost all of the_Sterne strain of Bacillus anthracis remained as vegetative cells. [__Jopined eon not recall any details of their morphology. The fermenter contents were subsequently pumped to a centrifuge. The supernatant was collected for further protein isolation and purification. The pellet of vegetative cells of the Sterne strain of Bacillus anthraci discarded. La could not recall if fermented th strain of Ba@ilius anthracis; however, reiterated never fermented the Ames strain of Bacillus anthracis. advised fermentation of virulent bacterium, such as the Ames strain of Bacillus anthracis, was a very dangerous rocess as the fermenter itself was an excellent aerosolizer. dvised even although the air used during the fermentation process was continually channeled from the fermenter to an : incinerator, thereby destroying any aerosolized ‘bacterium, to produce virulent spores, such as the Ames strain of Bacillus anthracis, one would likely contaminate the entire suite and place his fellow co-workers at risk. Using the fermenter at USAMRIID with the Ames strain Bacillus anthracis was specifically forbidden. .[L___Jopinea the elemental signature of biological agents might provide clues to methods of production. For example, Bacillus anthracis spores that were grown on agar plates ought to contain trace elements unique to the agar. For small scale Bacillus anthracis spore preparations, the _agar plate method is the preferred method for spore production. perfected this procedure. Po noted the agar needed a quantity of Magnesium (Mg) to be present to assist in Bacillus anthracis sporulation. [__jadvised for large scale Bacillus anthracis spore production, such as that found in the former Russian offensive biological weapons program, a fermenter was used. The fermenter method would thus not show trace elements unique to the agar. Ifa Silicon anti-foam was utilized, then the Silicon may be a trace