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FD-302a (Rev. 10-6-95) bé b7¢ 279A-WE-22293 6-DUGWAY Continuation of FD-302 of | | ,On.12/07/2006 _ , Page 3 vials of seed stock originally sent Z Ivins in 1997. L___] identifies picture #72 as beads made from IVINS' original spore stock in November, Or another Dugway- USAMRIID bulk spore growth project. [ identifies the tube in picture #74 labeled "B. ant Ames" as an original March, 1997 seed stock tube ambiguously labeled by IVINS. additionally identified the tubes in pictures and 75 as identically labeled March 1997, IVINS seed stock. [___aescribea the process by which[]produced the beads described in picture #72 as streaking spores for isolation, picking 3-5 colonies, and adding them to the bead storage solution. confirmed that only one tube of this original stock was used in the 1997 production runs that contributed to RMR 1029. L[___| explained that ould steak for isolation on either Blood Agar Plates (BAPs) or Tryptic Soy Agar (TSA), pick multiple, three (3) to five (5), colonies, resuspend and used to inoculate BAPs for confluent growth. BAPs were then used to inoculate the Leighton-Doi_ media contained in a glass fermentation vessel.[__—_—iJ always picks more than one colony culation in order to assure a heterogenous population. added Antifoam A to the media before sterilization did not control for pH during fermentation of Ba. elieves that controlling for pH is unnecessary in that a natural rise (basic), fall (acidic), and rise (basic) profile is a natural process in the cellular life cycle. [ ___] described the Bg contamination of 1997 Dugway- USAMRIID batches as either environmental contamination of the count plates, post fermentation (Suitable to send on to USAMRIID), or contamination within the fermentation (not suitable. to send on to USAMRIID). Unsuitable fermentation runs were destroyed by autoclaving.[_§______]described the noted 2003 Bacillus subtilis (Bs) contamination as one in the same, or interchangeable, with the Bg contamination de i rlier as easily distinguished custard orange colonies. has never noted a mucoid/contamination colony in any fermentation run. | oes unable to explain, based on[ _] notebook entries an testing any difference between fermentation production runs dated 09/09/1997 and 09/15/1997 (the 7th lot) which were described by IVINS to be "too dirty" to be added to RMR 1029. [_—j proceeded with one additional fermentation run