◆ SpookStack

FD-302a (Rev? 10-6-95) 279A-WF-222936| _—| 302 . Continuation of FD-302 of BRUCE EF. IVINS ,On 02/12/2003 __,Page 3 The Dugway material has been kept in the cold room in B3 since it was received in 1997. There is a record of how much material was initially received as well as how much has been removed and where it went. IVINS thinks that all of the Dugway material has been stored with his lab group and kept in the two original flasks it was placed in. Each flask initially had 500 milliliters of wet spore preparation, and only one flask of material remains. IVINS advised that 1 milliliter or less of the Dugway material could be taken and it wouldn't be missed. Double purified material needs to have a chromatogram done to determine whether it was run through a gradient. A trace of the gradient would remain on the material and should be indicated by a peak on the chromatogram. Some possible gradients include sucrose, hypaque, renografin (which became known as renocal but may not be manufactured any longer), or phycoll. IVINS did the following calculation estimates to determine how much of the Dugway spores would have been missing if they had been used in the anthrax mailings. 2.0-2.5 grams of material were present in each letter with a 10°/mL spore concentration. In order to achieve that spore concentration, 80-100L of runs would have been necessary and approximately 300mL of the Dugway material would have been required. This amount of missing material would have been noticed. IVINS thinks that most of the people at USAMRIID use broth to grow B.a., although he can't say for certain how everyone else there grows their spores. Although B.a. can be grown on agar, nicer spores are produced in broth, and they can be purified more easily and are "hotter". If one needs to purify more than a very small amount of B.a., it is difficult to use agar as you would need numerous plates. Spores grown up on agar are dirtier than broth grown. IVINS does not know if this is because the spores pick up debris from the agar or if there are media components on the surface of the spore. No matter how agar grown spores are washed, some agar always remains on the spores. The protocol that IVINS used to grow B.a. is the same one published in the paper titled "Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin". bé IVINS,[ _fand several other USAMRIID scientists co-authored bIc