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FD-302a (Rev. 10-6-95) 279A-WF-222936, 279A-BA-C101392 Continuation of FD-302 of [Lt Ci‘CUY ,On 01/19/2002 , Page 3 b c (U)Much of the work at USAMRIID involved growing spores and placing them in suspension in injection vials. The number of vials was not normally counted. Someone with access could easily remove a sample and not be detected. Samples of that nature in vials could remain stable indefinitely. These vials were used_to challenge the vaccine_in animals at USAMRIID worked with BRUCE IVINS, The test animals would either be injected or exposed to aerosol. All aerosol work was “wet” and performed by the Aerosolization Division. No “dry” spores were manufactured or used. (U) Once a virulent sample was obtained, it could be placed into flask(s) of sporulation media, or any liquid media that is nutrient-deficient. As the bacteria grew, the food supply would run out and sporulation would begin. If the flask(s) were left to sit for a few days, an enzyme would be produced that would sluff off unneeded parts of the cells. At that point in the process, the spores would be crude, but effective. (U) The next step would involve gently spinning the suspension and washing it several times. It could be washed simply by adding water and spinning to remove supernatant material such as the media and cell debris. This process would leave whitish pellets. The pellets would be put into a vortex, then samples could be removed. The process would result in white, refractile spores. The concentration could then be calculated. The spores could be put in a 1:10 solution, then the solution process could be repeated until individual colonies could be identified under a microscope. The count of the colonies, adjusted for the number of dilution steps, would reveal the concentration of the spores. (U) The spores could be converted to a weaponized powder by one of two methods. One is lyophilization, or freeze-drying. The sample is frozen and a vacuum is applied. Under a vacuum, ice sublimates, or changes from solid directly to gas. Most bacteria can be freeze-dried and often are to make them more stable. However, B. anthracis spores are extremely stable and do not require the protection of freeze-drying. _ OS The other method involves spinning the sample down and resuspending it in organic solvent (not water). A film material such as bentonite or fumed silica could be added at this point to make a free-flowing powder. This material could also be introduced to freeze-dried spores. A German firm named DEGUSSA manufactures several types of fumed silica under the brand name