CASE CLOSED … what really happened in the 2001 anthrax attacks?

* USAMRIID today provided under FOIA the reports attached to Dr. Ivins’ June 26, 2001 email – they are a series of technical studies on the effect of storage conditions on counts in suspension

Posted by Lew Weinstein on December 7, 2011

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2 Responses to “* USAMRIID today provided under FOIA the reports attached to Dr. Ivins’ June 26, 2001 email – they are a series of technical studies on the effect of storage conditions on counts in suspension”

  1. DXer said

    Batch 29 has now been restored. Thx again to USAMRIID FOIA for contacting the USAMRC webmaster about the glitch.

  2. DXer said

    Kudos to USAMRIID FOIA for its sometimes record-breaking efficiency. These reports were sent after the problem arose at Battelle earlier that month about the foaming and discussion of the perceived need for an antifoaming agent. It was determined that the foaming was due to the age of the spores.

    As I previously have discussed, upon reformatting of the webpage, the webmaster at USAMRC that handles the USAMRIID FOIA webpage inadvertently has not uploaded (the previously uploaded) Batch 29. The batch contains almost all the emails about the use of silica as an antifoaming agent by Battelle — necessary because the old spores tended to foam a lot.

    The contents of Batch 29 are now missing.

    https://mrmc.amedd.army.mil/index.cfm?pageid=foia_reading_room.overview#

    USAMRIID FOIA/the webmaster should re-upload them so that GAO has the benefit of these important emails.

    In the meantime, I can provide most of them here.

    Sent: Monday, June 04, 2001 5:06 PM
    To: ‘Bruce.Ivins@DET.AMEDD.ARMY.MIL‘
    Subject: Spores and Foaming

    Bruce,

    thought it would be easier if I contacted you directly. With regard to the foaming issue.

    When I go back to the original suspension you sent in the 50 ml conical tube and vortex it the same foaming phenomenon occurs. So I do not believe it is a glassware problem or washing problem.

    If you will/could go back to one of your 10E10 stocks of the same spore prep. And also make a 10E9 dilution and vortex it to see if you get the same thing.

    As described before, it’s like whipped cream on top of the water and will not go back into suspension unless it sits for a day or more. When I enumerate the suspension under the whipped cream it is 0.5-1 log lower than what is expected (i.e. what should be 10E9 is 5 x 10E8 to 1 x 10E8).

    In the mean time do you have any ideas on a defoaming agent?

    *** REDACTED ***

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Bcc:
    Subject: RE: Spores and Foaming
    Date: Tuesday, June 05, 2001 7:29:36 AM
    When we mix the spores at that concentration, we don’t vortex. I should have said that. I think the
    reason that it may foam is that the spore suspension is so pure. In the Vollum 1B spore suspension from
    1965 which is about 4 – 5 X 10^10 per ml, and MUCH dirtier, there is no foaming upon vortexing. The
    spores you have were twice purified with Hypaque gradient centrifugation. The spores are very
    hydrophobic, I believe. I suppose you could try to add a little Tween 80 to the spores to see if that
    helps. I’ve heard that in the “OLD DAYS” back in the 50s and 60s here at Detrick, they would soemtimes
    add a little Tween 80 to the spores to be aerosolized. We haven’t added any because we didn’t want to introduce another variable into the challenge. If you add something else to the spores being aerosolized,

    you may have to be able to demonstrate that the “anti-foam” has no effect on spore LD50, the infection
    process, or the specific immune response to the infection. As I said, when we mix the spores at that
    concentration, we just rock back and forth or gently swirl. If you want to add something to the spores
    before challenge, I think you should first run it by the IPT for their comments.
    – Bruce

    —–Original Message—–
    From:
    Sent: Monday, June 04, 2001 5:06 PM
    To: ‘Bruce.Ivins@DET.AMEDD.ARMY.MIL’
    Subject: Spores and Foaming
    Bruce,
    thought it would be easier if I contacted you directly. With regard to
    the foaming issue. When I go back to the original suspension you sent in
    the 50 ml conical tube and vortex it the same foaming phenomenon occurs. So
    I do not believe it is a glassware problem or washing problem. If you
    will/could go back to one of your 10E10 stocks of the same spore prep. and
    also make a 10E9 dilution and vortex it to see if you get the same thing.
    As described before, it’s like whipped cream on top of the water and
    will not go back into suspension unless it sits for a day or more. When I
    enumerate the suspension under the whipped cream it is 0.5-1 log lower than
    what is expected (i.e. what should be 10E9 is 5 x 10E8 to 1 x 10E8). In the
    mean time do you have any ideas on a defoaming agent?

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Bcc:
    Subject: RE: Spores and Foaming
    Date: Tuesday, June 05, 2001 9:04:29 AM
    , these spores are exactly the same spores used for the other rabbits for BioPort. We do get some
    foaming, but still get a high dose with 3 X 10^9 per ml. Would it help to use more suspension in your
    container? We’ve used these spores for quite awhile with success. As I said, maybe it’s because they
    are so clean that they clump. If there were some cell material stuck to the outside, perhaps they would
    perform a little more like the old Vollum 1B spore suspension or like some of the less pure Ames spore
    suspensions we have used in the past.
    – Bruce

    —–Original Message—–
    From:
    Sent: Tuesday, June 05, 2001 8:24 AM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Cc:
    Subject: RE: Spores and Foaming
    Bruce,
    One other question. Is were the spores that you sent to us prepared the
    same way as the ones RIID used on the BioPort rabbit studies or the same
    spore prep.? Or did you use different AMES spores? Looks like even though
    I’m a log low on the AGIs than expected I can still hit the targeted LD50
    range and will use These spores and mix by inversion. Thanks for answering
    my questions

    —–Original Message—–
    From: Ivins, Bruce E Dr USAMRIID [mailto:Bruce.Ivins@DET.AMEDD.ARMY.MIL]
    Sent: Tuesday, June 05, 2001 7:30 AM
    To: ‘
    Subject: RE: Spores and Foaming
    , When we mix the spores at that concentration, we don’t vortex. I should
    have said that. I think the reason that it may foam is that the spore
    suspension is so pure. In the Vollum 1B spore suspension from 1965 which is
    about 4 – 5 X 10^10 per ml, and MUCH dirtier, there is no foaming upon
    vortexing. The spores you have were twice purified with Hypaque gradient
    centrifugation. The spores are very hydrophobic, I believe. I suppose you
    could try to add a little Tween 80 to the spores to see if that helps. I’ve
    heard that in the “OLD DAYS” back in the 50s and 60s here at Detrick, they
    would soemtimes add a little Tween 80 to the spores to be aerosolized. We
    haven’t added any because we didn’t want to introduce another variable into
    the challenge. If you add something else to the spores being aerosolized,
    you may have to be able to demonstrate that the “anti-foam” has no effect on
    spore LD50, the infection process, or the specific immune response to the
    infection. As I said, when we mix the spores at that concentration, we just
    rock back and forth or gently swirl. If you want to add something to the
    spores before challenge, I think you should first run it by the IPT for
    their comments.
    – Bruce

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Bcc:
    Subject: RE: Spores and Foaming
    Date: Tuesday, June 05, 2001 9:20:04 AM
    ,
    We usually spray at a concentration of 3 X 10^9 per ml. That gives us an aerosol inhaled dose of about
    100- 200 LD50s in a 10-minute spray. You can try a test run with some Tween 80 and see if that helps.
    I seem to recall they used some concentration between 0.01% and 1%, but I don’t remember exactly,
    since it was given to me by word-of-mouth. I still recommend getting the IPT’s opinion. If there’s no
    other way to aerosolize than using anti-foam, you may have to do so, but I would hesitate to do it
    unless absolutely necessary.
    – Bruce

    —–Original Message—–
    From:
    Sent: Tuesday, June 05, 2001 8:02 AM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Spores and Foaming
    True but when I nebulize a 10E9 conc. the foaming happens. Do you not
    nebulize that high of a conc.? Also even at lower dilutions my AGI
    enumerations are approx. 1 log lower than what I expect. Thus I appears
    that even al low conc. they foam out of suspension and I’ll have to add some
    type of defoaming agent.

    From:
    Sent: Tuesday, June 05, 2001 9:32 AM
    To: ‘
    Cc: ‘bruce.ivins@det.amedd.army.mil’
    Subject: RE: Spores and Foaming
    : I believe we are resolving our questions regarding the foaming and
    we won’t be vortexing anymore. Bruce has helped us out immensely (see
    below). Could you please provide information regarding the anti-foam
    formulation that your staff uses – if any – for these high concentration
    anthrax aerosols.
    Thanks

    —–Original Message—–
    From:
    Sent: Tuesday, June 05, 2001 9:22 AM
    To:
    Subject: FW: Spores and Foaming
    —–Original Message—–
    From: Ivins, Bruce E Dr USAMRIID [mailto:Bruce.Ivins@DET.AMEDD.ARMY.MIL]
    Sent: Tuesday, June 05, 2001 9:20 AM
    To: ‘
    Subject: RE: Spores and Foaming
    We usually spray at a concentration of 3 X 10^9 per ml. That gives us an
    aerosol inhaled dose of about 100- 200 LD50s in a 10-minute spray. You can
    try a test run with some Tween 80 and see if that helps. I seem to recall
    they used some concentration between 0.01% and 1%, but I don’t remember
    exactly, since it was given to me by word-of-mouth. I still recommend
    getting the IPT’s opinion. If there’s no other way to aerosolize than using
    anti-foam, you may have to do so, but I would hesitate to do it unless
    absolutely necessary.
    – Bruce

    —–Original Message—–
    From:
    Sent: Tuesday, June 05, 2001 8:02 AM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Spores and Foaming
    True but when I nebulize a 10E9 conc. the foaming happens. Do you not
    nebulize that high of a conc.? Also even at lower dilutions my AGI
    enumerations are approx. 1 log lower than what I expect. Thus I appears
    that even al low conc. they foam out of suspension and I’ll have to add some
    type of defoaming agent.

    From:
    Sent: Tuesday, June 05, 2001 9:57 AM
    To: Ivins, Bruce E Dr USAMRIID
    Cc:
    Subject: RE: Spores and Foaming
    We need to look at your spray factor and adjust accordingly – we do NOT want to change anything
    from what we do here – I know Bruce is being helpful – BUT——
    Can I see the numbers and starting conc. etc.????
    Thanks,

    From:
    Sent: Tuesday, June 05, 2001 8:02 AM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Spores and Foaming
    True but when I nebulize a 10E9 conc. the foaming happens. Do you not
    nebulize that high of a conc.? Also even at lower dilutions my AGI
    enumerations are approx. 1 log lower than what I expect. Thus I appears
    that even al low conc. they foam out of suspension and I’ll have to add some
    type of defoaming agent.

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: Spores
    Date: Tuesday, June 05, 2001 1:49:25 PM
    Attachments:
    Hi,
    I reworded the Statement of Work for the anthrax spores according to what you said. I’ve
    enclosed the file here. Please let me know if this looks OK. When it meets your OK, I’ll send it over to
    Thanks, and see you in Annapolis!!
    – Bruce

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: RE: Spray factor data
    Date: Wednesday, June 06, 2001 12:54:43 PM
    – Does this mean that you and perhaps others (me? ? etc.?)are headed to Battelle to work on
    the spore/aersol/foaming/clean or dirty glassware problem? Let us know!
    – Bruce

    —–Original Message—–
    From:
    Sent: Wednesday, June 06, 2001 12:08 PM
    To:
    Cc: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Spray factor data
    Enclosed is a representative sample of what I typically see. Are these
    spray factors more in line with what you see or are they still not as
    efficient? Some time during the conference can you, myself and
    get together to discuss this? Also, thought it might be worth
    while if after the conference on Wed. or the IPT on Friday if it might be
    possible for me to come to USAMRIID and see if your spores look similar
    (they should) and react the same why after making a 10E9 suspension and
    vortexing and a 10E9 suspension then nebulizing to see if the foaming
    occurs. Assuming you have the time and it is allowable. As you saw from
    the spay factor calc. you determined, I can not achieve the targeted aerosol
    conc. to reach 100-200 LD50s for the BioPort study.

    —–Original Message—–
    From:
    Sent: Wednesday, June 06, 2001 10:23 AM
    To: ‘
    Subject: RE: Spray factor data
    Attached to spread sheet is my recalculated spray factors – the way we
    calculate etc.
    By my calculations – an aerosol concentration of around 5 X 10(6) cfu/l is
    needed for the animals to get around 150 LD50s
    Your spray factors are in the low range compared to ours – we usually get
    better efficiency
    Are you going to repeat this???

    —–Original Message—–
    From:
    Sent: Tuesday, June 05, 2001 4:24 PM
    To:
    Cc:
    Subject: Spray factor data
    Here is the spray factor information that you have been waiting for.
    Limited reps. because we had the foaming problem. It looks like I would
    have to start with a neb. conc. of approx. 2.9 X10E9 to hit 100-200 LD50s.
    Do you usually a larger drop in AGI conc. from the initial neb. 10E9
    concentration as compared to the lower dilutions?

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: Animal protocol
    Date: Wednesday, June 06, 2001 2:22:59 PM
    Attachments:
    Here is an animal protocol for submission to the LACUC.
    Thanks!
    – Bruce

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: RE: – Stability Indicating Assay sub-team
    Date: Thursday, June 07, 2001 8:06:45 AM
    Hi,
    telephone number and email address are:
    I think that you will find him a very competent and knowledgeable individual, with a
    great deal of personal experience with respect to fermentation, production of antigen, adsorption onto
    Alhydrogel, analysis of antigen product, and desorption from Alhydrogel.
    Sincerely,
    Bruce Ivins

    —–Original Message—–
    From:
    Sent: Thursday, June 07, 2001 7:52 AM
    To: ‘Bruce.Ivins@DET.AMEDD.ARMY.MIL’
    Subject: – Stability Indicating Assay sub-team
    Dear Bruce,
    I am wondering if you could provide me with telephone number and his
    E:mail address so that I can contact him. I do not have USAMRIID directory
    with me.
    Regards,

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: RE: Suggestions for study
    Date: Wednesday, June 13, 2001 5:21:39 PM
    Sure, When would you like the material? How many doses of each? Should I drive it down to
    you, or is there someone here that can get it to you?
    Regards,
    – Bruce

    —–Original Message—–
    From:
    Sent: Monday, June 11, 2001 11:31 AM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Suggestions for study
    Dear Bruce,
    The monkeys are arriving for the anthrax study. Any chance I can get the
    vaccines (both the new and the old) from you to start the immunizations?

    —–Original Message—–
    From: Ivins, Bruce E Dr USAMRIID [mailto:Bruce.Ivins@DET.AMEDD.ARMY.MIL]
    Sent: Thursday, May 31, 2001 3:18 PM
    To:
    Subject: RE: Suggestions for study
    I forgot to add that I wasn’t sure of the final approved groups for
    vaccination, and if some of the groups received reduced levels of PA.
    – Bruce

    —–Original Message—–
    From:
    Sent: Thursday, May 31, 2001 2:32 PM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Suggestions for study
    Dear Bruce,
    The ACUC has approved our anthrax vaccine study in rhesus macaques. We
    should be getting the animals in within a month. We have approval to test
    the effect of our CpG ODN on both the old and new vaccines, if you can
    provide them for immunization.
    Hooray.

    —–Original Message—–
    From: Ivins, Bruce E Dr USAMRIID [mailto:Bruce.Ivins@DET.AMEDD.ARMY.MIL]
    Sent: Friday, May 11, 2001 7:58 AM
    To:
    Subject: RE: Suggestions for study
    Hi,
    I’ll be happy to provide you with the information. The current,
    FDA-approved human anthrax vaccine consists of supernatant material,
    primarily anthrax protective antigen (in undetermined and varying amounts),
    from fermentor cultures of a toxinogenic, non-encapsulated strain of B.
    anthracis, V770-NP1-R. Each 0.5-ml dose of the vaccine delivers about 0.725
    mg of metallic aluminum (from the aluminum hydroxide adjuvant), protective
    antigen in the range of about 0.5 to 20 micrograms, and other material such
    as lethal factor, some edema factor (in certain lots but not others) and
    other cellular material. The proposed new vaccine will contain less aluminum
    (0.5 mg/dose), no lethal factor, no edema factor, and no other B. anthracis
    material other than a specified and constant, defined amount of protective
    antigen. We are tentatively targeting 50 micrograms as that amount. Use of
    the proposed new vaccine in rabbits and rhesus macaques has demonstrated
    efficacy against challenge, but has not demonstrated any observable
    morbidity, mortality, or local or systemic reactogenicity. (Formal toxicity
    studies are scheduled, but have not been conducted yet.) Thus, there is good
    evidence of protection, but no evidence of adverse reactions associated with
    the product. I should point out that the original vaccine contains
    formaldehyde, which may contribute to some of the reactogenicity seen in
    humans with the current vaccine. The vaccine you will be testing will not
    contain formaldehyde. We have not yet published our findings with respect to
    toxicity/reactogenicity of the new vaccine. Unfortunately, the studies
    describing efficacy of the new vaccine are in abstract form, but have yet to
    be put into a formal publication. I would suggest the following references
    are pertinent to the concerns of your ACUC:
    1. Comparative efficacy of experimental anthrax vaccine candidates
    against inhalation anthrax in rhesus macaques. B. E. Ivins, M. L. M. Pitt,
    P. F. Fellows, et al. 1998. Vaccine 16:1141-1148.
    2. Comparative efficacy of a recombinant protective antigen vaccine
    against inhalation anthrax in guinea pigs, rabbits, and rhesus monkeys. M.
    L. M. Pitt, B. Ivins, J. E. Estep, et al. 1996 Abstracts of the Annual
    Meeting of the American Society for Microbiology. E-70, p. 278.
    3. Comparison of the efficacy of purified protective antigen and
    MDPH to protect non-human primates from inhalation anthrax. M. L. M. Pitt,
    B. E. Ivins, J. Farchaus and A. M. Friedlander. 1996. Salisbury Medical
    Bulletin Special Supplement, # 87, p. 130.
    I hope this has been helpful.
    – Best regards,
    – Bruce
    —–Original Message—–

    From:
    Sent: Thursday, May 10, 2001 3:54 PM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Suggestions for study
    Dear Bruce,
    Hope all is well.
    Our protocol was reviewed this morning by the ACUC. They’ve tentatively
    approve it, with the caveat that I need to provide more background on the
    safety of the vaccine immunogen. Specifically, they want to know about the
    PA-based vaccine as well as the original vaccine (which I hope to include as
    a positive control). What can you tell me about the safety of these agents?
    Can you provide some background on how they are manufactured, how pure they
    are, and what other studies have been done that support going into monkeys?
    All the best,

    From: Ivins, Bruce E Dr USAMRIID

    To:
    Subject: RE: Suggestions for study
    Date: Wednesday, June 13, 2001 5:44:16 PM
    ,
    We usually make up the vaccine for use no more than about 2-3 days ahead of schedule,
    although we’ve not done the stability studies to see whether we can go longer or not. (My guess is that
    we could. I just don’t.) Rather than ship it, I would rather carry it down to you on gel ice in person.
    That way, nothing could happen en route with the third-party shipper/handler. I could get it down to
    you the first part of next week if needed, or later if desired. I’ll get you extra amounts of each vaccine.
    It’s no problem to make the vaccine, and it will only take a couple of hours to drive down, give it to
    you, and come back. I’m quite excited about the experiments.
    – Bruce

    From:
    Sent: Wednesday, June 13, 2001 5:37 PM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Suggestions for study
    Dear Bruce,
    I believe the protocol calls for a prime/boost, just as planned for use in
    humans. We will have 10 animals/arm, and thus need 20 doses each of the new
    vaccine and the old vaccine. As I recall, they are already in alum, ready
    for administration. We’ll just add our ODN and go. Naturally, we need a
    bit extra since there’s some wastage.
    In terms of timing, that depends on how stable the vaccine is. I assume its
    made up in advance and stored in the fridge? If so, we could accept it
    immediately, and start the injections as soon as we can get on the animal
    handler’s schedule. If it’s perishable, we’ll have to plan 5the timing and
    then let you know. In terms of getting it here, can you ship it on ice?
    Seems a shame to make you drive all the way down. If you or a colleague are
    coming this way, we’d be happy to wait or a hand delivery.
    Let me know, or perhaps we should chat by phone?
    —–Original Message—–

    From: Ivins, Bruce E Dr USAMRIID [mailto:Bruce.Ivins@DET.AMEDD.ARMY.MIL]
    Sent: Wednesday, June 13, 2001 5:22 PM
    To: ‘
    Subject: RE: Suggestions for study
    Sure, When would you like the material? How many doses of each?
    Should I drive it down to you, or is there someone here that can get it to
    you?
    Regards,
    – Bruce

    —–Original Message—–
    From:
    Sent: Monday, June 11, 2001 11:31 AM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Suggestions for study
    Dear Bruce,
    The monkeys are arriving for the anthrax study. Any chance I can get the
    vaccines (both the new and the old) from you to start the immunizations?
    —–Original Message—–

    From: Ivins, Bruce E Dr USAMRIID [mailto:Bruce.Ivins@DET.AMEDD.ARMY.MIL]
    Sent: Thursday, May 31, 2001 3:18 PM
    To:
    Subject: RE: Suggestions for study
    I forgot to add that I wasn’t sure of the final approved groups for
    vaccination, and if some of the groups received reduced levels of PA.
    – Bruce
    —–Original Message—–

    From:
    Sent: Thursday, May 31, 2001 2:32 PM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Suggestions for study
    Dear Bruce,
    The ACUC has approved our anthrax vaccine study in rhesus macaques. We
    should be getting the animals in within a month. We have approval to test
    the effect of our CpG ODN on both the old and new vaccines, if you can
    provide them for immunization.
    Hooray.

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: Thanks again!
    Date: Thursday, June 14, 2001 6:17:29 AM
    I just wanted to tell you once again how much we appreciate all your efforts on the 4th
    International Conference on Anthrax. We enjoyed working with you both immensely. The comments that
    we heard from many other attendees point to the meeting having been a great success, in large part
    due to you. You are both very competent and very personable, and you are a credit to the ASM. Take
    care, and many thanks again!
    Sincerely,
    Bruce Ivins
    Research Microbiologist
    USAMRIID Bacteriology Division

    From: Ivins, Bruce E Dr USAMRIID
    To: Ivins, Bruce E Dr USAMRIID
    Cc:
    Subject: RE: Spores and Foaming
    Date: Friday, June 15, 2001 12:16:15 PM
    ,
    I am sending you on Monday, 3 15-ml polypropylene tubes of Ames spores, each of which
    contains about 10-11 ml of spores at 3.9 X 10^10 per ml. Here are suggestions as to how to handle
    them to minimize foaming. This is how we handle them, by the way.
    1) To resuspend the spores, don’t shake or vortex the tube. Instead, GENTLY tip the tube back
    and forth until the spores are suspended. If spores are in a bottle or flask, then you can GENTLY swirl
    to resuspend the spores.
    2) We dilute the spores 1:13 (1 ml spores into 12 ml Sterile water for injection) for spraying
    rabbits. I would suggest taking spores not from the very top of the tube and adding them to water. To
    mix the new suspension (about 1.3 X 10^9 per ml) gently tip or swirl the container.
    3) Before spraying, gently tip or swirl the spore suspension before gently pouring into the collison.
    If you have any questions, please call me at . If you are still having technical problems
    with the spores you should get next Tuesday, please let me know. will come up the following
    week (what day is best for you?) If you think my presence would be valuable, let me know, and I’ll also
    come. Otherwise, it will just be her. (I don’t mind coming at all – I would just like my presence there to
    serve a useful purpose, and not be just a warm body.)
    – Bruce

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: RE: Spores and Foaming
    Date: Friday, June 15, 2001 12:26:09 PM
    We shock the dilution that we are going to spray.
    – Bruce

    —–Original Message—–
    From:
    Sent: Friday, June 15, 2001 12:20 PM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Spores and Foaming
    Thanks,
    When you heat shock do you shock the stock or the dilution or both?

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: RE: Spores and Foaming
    Date: Friday, June 15, 2001 12:28:38 PM
    OK,
    Again, let and me know about whether or not she, or both of us need to come up the week
    after next.
    – Bruce

    —–Original Message—–
    From:
    Sent: Friday, June 15, 2001 12:22 PM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Spores and Foaming
    Thanks! I’ll let you know what happens next week.
    —–Original Message—–
    From: Ivins, Bruce E Dr USAMRIID [mailto:Bruce.Ivins@DET.AMEDD.ARMY.MIL]
    Sent: Friday, June 15, 2001 12:26 PM
    To: ‘
    Subject: RE: Spores and Foaming
    We shock the dilution that we are going to spray.
    – Bruce

    From:
    Sent: Friday, June 15, 2001 1:01 PM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: Spores and counting
    Dr. Ivins,
    A question on how you enumerate. Our SOPs say a plate should contain from
    25-250 spores per plate (we do 5 plates per dilution). Do you have criteria
    for rejecting low or high numbers? Say I get a plate that has 12 colonies
    and all remaining plates are within the 25-250 range. Do you reject that
    plate and average the 4 remaining, use all 5 and average, reject all 5 and
    re-enumerate with 5 more etc. I ask this, because it could potentially be a
    GLP issue. I apologize if this is any SOP that you have sent but
    I have not seen them yet.

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: RE: Spores and counting
    Date: Friday, June 15, 2001 2:08:24 PM
    We don’t have a specific number. When we are counting AGI’s, after plating, we put the samples back
    into the cold until the next day. We examine all of the plates. If one group is contaminated or out of
    range, we will go back and redilute and replate from the AGI sample. We have had to do that only a
    handful of times out of thousands of samples. We usually do AGIs at 3 per set, 10-4 and 10-5 dilutions
    (3 plates for each dilution). If you are getting low counts, you might do 10-3 and 10-4 dilutions. If we
    get at least 2 of 3 readable plates, we go ahead and count the set and average the counts.
    – Bruce

    —–Original Message—–
    From: ]
    Sent: Friday, June 15, 2001 1:56 PM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Spores and counting
    How many have to be low or high before you reject the whole set?
    —–Original Message—–
    From: Ivins, Bruce E Dr USAMRIID [mailto:Bruce.Ivins@DET.AMEDD.ARMY.MIL]
    Sent: Friday, June 15, 2001 1:20 PM
    To: ‘
    Subject: RE: Spores and counting
    We count 15 – 150 colonies per plate. Because of the large size of the
    colonies, it’s next to impossible to accurately count over 150 colonies on a
    single plate. I have told many times that 15 – 150 is a more
    realistic value than 25 – 250 or 30 to 300. If one plate is over count,
    under count, or contaminated, we take note of it and disregard it in the
    count and averaging.
    – Bruce

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: FW: Spores
    Date: Monday, June 18, 2001 11:18:57 AM
    Attachments:
    Hi,
    I’m sending this to you again. I just wanted to make sure you received it. When I hear from you
    about it, I’ll send it forward here. I think if you send us material about every 2-4 weeks, that would be
    good. That will give us the time to purify each batch. Hope you enjoyed Annapolis! I thought there
    were some good talks there.
    – Bruce

    From: Ivins, Bruce E Dr USAMRIID
    To: ” Ivins, Bruce E Dr USAMRIID
    Cc:
    Subject: RE: Spores and counting
    Date: Monday, June 18, 2001 1:33:51 PM
    ,
    The spores were sent to this morning by Federal Express. Please let me know when you
    get them. Also, keep me posted on your tests with them, so, if we need to, we can plan a trip up there
    for next week. Remember, avoid vortexing and vigorously shaking them. If it seems as if the spores I
    sent you are clumped a bit at the top, then take the spores you need from about midway down into the
    suspension after you resuspend them.
    – Bruce

    From: Ivins, Bruce E Dr USAMRIID
    To: Ivins, Bruce E Dr USAMRIID
    Cc:
    Subject: RE: Spores and counting
    Date: Monday, June 18, 2001 1:33:51 PM

    The spores were sent to this morning by Federal Express. Please let me know when you
    get them. Also, keep me posted on your tests with them, so, if we need to, we can plan a trip up there
    for next week. Remember, avoid vortexing and vigorously shaking them. If it seems as if the spores I
    sent you are clumped a bit at the top, then take the spores you need from about midway down into the
    suspension after you resuspend them.
    – Bruce

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: FW: Spores and counting
    Date: Monday, June 18, 2001 1:39:40 PM
    Hi, If Battelle still has trouble with the new spores I sent them this week, I think that there will be
    a trip up there next week, with definitely, probably – he wants to see the facilities where
    the rPA studies will be done – me maybe, and you if you’d like. I’ll let you know how things go.
    – Bruce

    From: Ivins, Bruce E Dr USAMRIID
    To:
    Subject: FW: – Stability Indicating Assay sub-team
    Date: Monday, June 18, 2001 4:14:22 PM
    Could you please include on your list of individuals to receive information about
    conference calls, IPT meetings, etc.? Thanks.
    – Bruce Ivins
    telephone number and email address are:

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