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FD-302a (Rev. 10-6-95) 279A-WF-222936-302 - bé . bic Continuation of FD-302 of fo EE 0 95/12/2006 , Page 2 growth media. RM, RMM, and CA consisted of organic salts and sugars, neither RM, RMM, nor CA contained unique elements such as the semi-metal Silicon (Si) . [___lopined should a growth media contain a unique element, then bacterium which propagate and/or sporulate_in the media would likely contain the unique element. However, [_____]advised[_]was not a sporulation expert. [| 2avised to knowledge the Ames strain of Bacillus anthracis was never recalled the Sterne strain of Bacillus anthracis for use in toxin studies. oreaee advised scientists at USAMRIID sought to understand the role ( of Edema Factor (EF), Lethal Factor (LF), and Protective entigen (PA) in anthrax pathogenesis, as well as discovery of their immunogenic properties. As such, there was a need to produce quantiti these purified EF, LF, and PA proteins via fermentation. [an Jexpiained the Sterne strain of Bacillus anthracis was use © accomplish this goal. [ —epinitially inoculated a 100 or 500 milliliter (ml) flask with the Sterne strain of Bacillus anthracis, after_a_period of time and with shaking, the*bacteria would propagate. a could not recall if transferred the Sterne strain of Bacillus anthracis from this ask to the fermenter via a simple pour, or if utilized a more aseptic procedure of injecting the flask Gontents, via_a_ sterile syringe, into the side port of the fermenter. was instrumental in determining the appropriate growth conditions. The pH of the media was crucial for Bacillus anthracis growth and was at a physiological relevant level. further advised the pH was neither strongly acidic nor caustic. The initial 100 or 500 ml flask of the Sterne strain of Bacillus anthracis rmented for an unknown period of time in the toxin studies. advised[__]simply could_not recall for how long the fermenter ran in the toxin studies. advised it would be possible to inoculate the 50 L fermenter with a smaller volume; , it would take much longer to obtain a reasonable yield. opined Bacillus anthracis generally doubled its population every two (2) hours in the 50 L fermenter. fermenter wou run overnight, morning the process was stopped as sufficient bacteria were present.