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TO; Washington Field From: Washington Field Re: 279A-WF-222936-USAMRIID, 07/14/2004 the[ sd medium was again inoculated, and the supernant from bic this solution was saved and given tol . Page 21 lists the[_______sdtvins medium recipe, On age 25, dated July 1, 1981, Ivins received from b6 es ee inoculated it into medium. After bic performing an experiment on it, Ivins concluded that all the v- bye 770-NPI-R strains at USAMRIID produce lethal anthrax toxin. On page 40, written August_21, 1981, Ivins compared toxin production byl in| ___] meaiur with slight shaking versus no shaking. He found, on page 41, that not as much growth was found with stationary cultures as with gently shaken cultures. Furthermore, Ivins determined that, for maximum toxin production in the[|___] medium, the inoculum blood plate must be incubated in an atmosphere containing carbon dioxide had been used in the past). On the same page, Ivins also found that, according to "Bergey's Manual of Determinative Bacterioloay". the maximum growth temperature of Ba was[__] On page 43, dated August 28, 1981, Ivins noted that there appeared to be differences with respect to growth of several avirulent and virulent strains of B. anthracis. He inoculated the Sterne, Vollum 1B, Ames, Colorado, and Texas strains into[____]medium and[ —_]mediun. On the next be page, Ivins determined that, when Inoculating from a plate, pve evidently the best growth and toxin production in R medium comes when a fresh (vegetative cells, not spores) blood plate incubated _in an atmosphere containing carbon dioxide is used. Page 60, dated October 3, 1981, detailed Ivins' observations that: b6 bIC bIF Incubation for carbon dioxide is better for the than incubation for only 3. The R medium oa en A medium for toxin production than the medium. 4. Toxin was bound to the heterologous protein (BSA) on the polypropylene centrifuge tube. . inoculum Ivins also wanted to demonstrate that carbon dioxide promotes toxin production when inoculum plates are incubated in carbon dioxide. Additionally, he wanted to determine the best times for incubation of the plates. After conducting an 4