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To: Washington i ® From: Washington Field Re: 279A-WF-222936-USAMRIID, 1/11/2006 ‘ chamber, so the amount of Ba was diluted in the AGIs compared to the starting Ba solution in the nebulizer. POST-CHALLENGE PROCESS: The post-challenge process was intended to determine the amount of pathogen or toxin actually breathed in by each animal. This determination was made by using information collected throughout the challenge (equipment settings, gauge readings, and animal respiration monitoring) and by determining the concentration of Ba collected into the AGIs by growing the Ba on agar plates and counting the colonies. Following an aerosol challenge, approximately 7 ml of the original Ba starting solution remained in the nebulizer. According to Ivins, it was normal procedure to autoclave the remaining material in the nebulizer prior to removing it from the hood at the end of the day. The technicians who actually carried out such tasks were not confident that this material was always autoclaved prior to removal from the hoodlines. The exterior of the AGIs was sprayed with a bleach solution prior to removal from the hood so the AGI contents could be plated to determine the concentration of Ba. Following an aerosol challenge, Ivins conducted most of the post-challenge work. Ivins was the PI for the majority of bé the anthrax studies, and he preferred to do his own post- bIC challenge work. [| sometimes assisted Ivins with the post- challenge work. The post-challenge plating to determine the concentration of Ba was conducted by creating serial dilutions of the collected samples in the The dilution tubes were kept pe in the walk-in cooler on the floor of Building because DIE there was not enough room in the refrigerator in room Several individuals stated that the 15 ml conical tubes containing the dilutions were disposed of after the post- challenge plating was completed, but other information suggested these tubes may have remained in the walk-in cooler on a long-