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Y ) . . © . FD-3024 (Rev. 10-6-95) 279A-WF-222936-USAMRIID Continuation of FD-302 of Bruce E. Ivins ,On 09/08/2004 __, Page 10 germination. IVINS first became aware of heat shocking back in the Gulf War. The Division Chief called IVINS in his office to discuss Are large NHP study. The Division Chief did not want the spores ao bUEF Renograffin purified because that was not was done in the old days. Spores used to be heat shocked SS This temperature was chosen because counts do no ecrease a Ls temperature and refractile spores are not killed. Spores that are used for intramuscular (IM) or aerosol challenges are heat shocked. All ‘spores are heat shocked in building 1412. IVINS does not know if anyone has studied virulence with respect to heat shock. However, did some studies on heat shocked material versus non-heat shocked material. IVINS noted that he told sen[ Taha Ses oo used Tween to break up a thick spore suspensions. who works for has added anti-foam to spore suspensions. IVINS then discussed the spore harvest procedure. Ames strain Ba, from a primary subculture, is streaked onto a sheep's blood agar (SBA) plate. Three "typical" colonies are selected and placed in phosphate buffer solution, then into Leighton and Doi broth for three days. Spores are harvested and run through a Hypaque density gradient. Hypaque is what is used now, where as Renocal or Renograffin is what was used in the past. IVINS learned this density gradient purification procedure from IVINS advised that he recently received an e-mail from USAMRIID command that stated the FBI was requesting all files, e-mails, etc. that related to the Ames strain of Ba. IVINS opined that this was a large undertaking and demonstrated this by showing the interviewing agents that just by searching the term "Ames" on his archived e-mails, he retrieved over 1200 e- mails. IVINS advised that he will be glad to provide whatever the FBI requested, but this request would require a large amount of time. Fifteen laboratory notebooks that were provided by IVINS were returned to him. An FD-597 was executed and detailed the numbers of the notebooks returned. IVINS was provided with a copy of the FD-597, and the remaining two copies are maintained in an FD-340, in the 1A section of the file.