◆ SpookStack

° , @ . ° ] “ - . » u we .. FD-302%(Rev. 10-6-95) 279AR-WF-222936-USAMRIID Continuation of FD-302 of BRUCE E. IVINS ,On 04 / 15 /2 003 , Page has knowledge of. It was turned in approximately three years ago for a new lyophilizer. For aerosol challenges, the stocks of Bacillus anthracis (B.a.) are kept in Suite B3. Dilutions are made and kept in large glass bottles. These bottles are placed in bags and then a transport can. They are transported to the Building 1412 airlock and placed in the refrigerator one to three days prior to the spray. The pre-challenge inoculum incubates overnight in Room| | 8) , the B.a. spores are heat shocked at cooled in ice, and placed in 9-10 mL tubes. One tube per animal is prepared. ‘The dilution is plated using Tryptic Soy Agar (TSA) from the Tissue Culture Group. As the challenge runs, the all glass impinger (AGI) water samples are collected after each run and given to IVINS to be plated. The colonies are counted the next day to determine the difference between the pre-challenge concentration and the dose that each animal actually received. The used agar plates are placed in a bag and autoclaved a few days later along with the rest of the spray trash. If a spray is conducted on a Tuesday or Wednesday, the plates are read the following day or two and clean up is on Friday. If it was a very busy week, clean up might wait until Monday. Plates, paper, and trash to be autoclaved are placed ina tidy sack, which is a large, extra thick paper bag. The individual's name and where the trash is from are noted on the sack and it is brought to the basement where the animal caretakers autoclave it. IVINS does not know how long the bag sits in the basement prior to being autoclaved. After a certain amount of time has passed after, being plated, B.a. colonies would no longer be able to be counted. After five to seven days, B.a. spores would form on any nutritional agar medium. Once the spores have formed, they are viable for a long time. There are still viable spores on the original B.a. Ames slant from Texas when it was last checked for the repository. Spores do not need nutrients because they are inert. Agar grown B.a. spores are almost always clumpier and dirtier and are not as high quality as broth grown spores.