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

* Ivins told the FBI in both April 2003 and April 2004 that he was sick that the material in the Fall 2001 anthrax mailings could have come from a stock made from the B.a. aerosol challenge trash in Building 1412

Posted by Lew Weinstein on September 29, 2015

The FBI has no case against this man ... but meanwhile he is dead and the real perpetrators are still out there.

The FBI has no case against this man … but meanwhile he is dead and the real perpetrators are still out there.

***

Aprilemailsabout1412trash-mod

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3 Responses to “* Ivins told the FBI in both April 2003 and April 2004 that he was sick that the material in the Fall 2001 anthrax mailings could have come from a stock made from the B.a. aerosol challenge trash in Building 1412”

  1. DXer said

    The wonderful USAMRMC officer who oversees FOIA has produced today an attachment titled “PRODUCTION, HARVEST AND PURIFICATION OF BACILLUS ANTHRACIS AMES SPORES FOR AEROSOL CHALLENGE.”

    http://mrmc.amedd.army.mil/content/foia_reading_room/Email%20Attachments/batch55_page_196_AMES.SPO.pdf

    This bears on Dr. Bruce Ivins’ frequently expressed concern that someone could have obtained seed stock from the trash in Building 1412 where the Ames aerosol challenges were conducted.

    US Attorney’s suggestion in August 2008 that only access in Building 1425 needed to be considered was always total crock. It was a huge mistake on which he premised his claim that Bruce Ivins was the culprit.

    The attachment I cut and pasted below is also at this link uploaded today.

    http://mrmc.amedd.army.mil/content/foia_reading_room/Email%20Attachments/batch55_page_196_UIB-BI-3_Redacted.pdf

    • DXer said

      I have always tried to target specific possibly relevant attachments for production — so as to avoid burdening USMRMC with time-consuming requests. The operation’s duties include, for example, dealing with medical-related requests from the families of soldiers who have lost their life.

      Perhaps USMRMC could provide — from among the attachments in the “attachments” folder but not yet uploaded — Amerithrax Inventory 1-13 and FBI receipts for property.

  2. DXer said

    The wonderful USMRMC official who oversees FOIPA provided this document that Ken Dillon had sought. It was an attachment to a March 19, 2004 email. I have copy and pasted the first document below.

    When I copy and paste things, I often inadvertently introduce formatting errors. With emails, it often happens because of redactions made pursuant to (b)(6). In the documents today, I believe there was only 1 redaction.

    Bacteriology Division Standing Operating Procedure
    SOP No.: UIB-BI-3 Effective date: 30 MAY 95 Page 1 of 6

    TITLE:Preparation of Bacillus anthracis Ames spores for aerosol challenge for Protocol # D94-09 – Efficacy of vaccination in protection of rhesus monkeys following aerosol infection with Bacillus anthracis: Dose titration of purified protective antigen (PA) combined with Alhydrogel.

    1. PURPOSE:
    To describe the preparation of the reference article, Bacillus anthracis Ames strain spores, for aerosol challenge of immunized rhesus monkeys.

    2. BACKGROUND:
    The procedure described is known to be suitable for producing and purifying spores of the virulent B. anthracis Ames strain for challenge. These spores have already been prepared and purified by the described procedure. There is approximately 125 ml of the spore suspension. The concentration of the spore suspension is about 1.40 X 1010 CFU per ml, as determined by enumerating colonies from dilutions of the suspension plated onto Tryptic Soy Agar. The purity, as judged by phase contrast microscopy, is greater than 99% unclumped, refractile spores, with less than 1% of the material being vegetative cells, clumped spores, non-refractile spores, vegetative cells, or debris. The spore batch is a combination of two similarly prepared batches (from a total of 8 runs of growth, spore harvest and purification), one prepared in OCT 94, the other prepared APR 95.

    3. PROCEDURE:

    Materials:

    1. Bacillus anthracis Ames, obtained by Gregory Knudson of the USAMRIID Bacteriology Division in 1980 from the U.S. Department of Agriculture, Ames Iowa. The strain was isolated from a cow which had died from anthrax. The strain is virulent, producing capsule, lethal toxin and edema toxin, and it is pXO1+, pXO2+ (Infect. Immun. 1990, 58:303-308). From the original slant obtained from the USDA, organisms were plated onto sheep blood agar in 1989. Colonies from the blood agar were used to inoculate Leighton and Doi medium (see below) and spores were harvested by centrifugation after three days growth. The spores were
    resuspended in brain heart infusion (Difco, Detroit, MI) containing 12% glycerol, and then frozen at -70OC in 1-ml freezer tubes in B-3. These tubes contain the seed stock of Ames spores used for production of spores for aerosol challenge. 2. Sterile water for injection, from McGaw, Inc., Irvine CA 92714-54895 (800/854-6851) lot # J350056A, EXP date DEC 95. This water is used for washing spores, making 1% phenolized water, and diluting Renografin-76.

    3. Distilled, deionized water, from the Milli-Q system in B-5, MMCN AO441, consisting of a carbon cartrdge, ion exchange cartridge and Organex-Q cartridge. The system runs off houses distilled water. This water is used for making all media (TSA, Leighton-Doi, sheep blood agar) and distilled water blanks for dilution plating for spore counts.

    4. Leighton and Doi medium, for both OCT 94 and APR 95 preparations, formulated as follows (J. Gen. Microbiol. 1982, 128:1591-1597):
    a) Difco nutrient broth powder (Difco Laboratories, P.O. Box 331058, Detroit, MI 48232-7058, (800)521-0851, catalog number 0003-01-6, Lot 24924, EXP DEC 97).
    b) Potassium chloride, 3.8 g, (Fisher Scientific Company, Chemical Manufacturing Division, Fair Lawn, NJ 07410, (800)766- 7000, catalog number P-217, Lot 714405, EXP DEC 99).
    c) Calcium chloride dihydrate, 0.6 g, (Fisher Scientific Co., catalog number C-79, Lot 770850, EXP DEC 99).
    d) Dextrose, 1.8 g, (Fisher Scientific Co., catalog number D16-500, Lot 912260A, EXP DEC 99).
    e) Magnesium sulfate heptahydrate, 5.0 g in 100 ml MilliQ H2O, (Fisher Scientific Co., catalog number M63-500, Lot 917414, EXP DEC 99).
    f) Manganese sulfate monohydrate, 0.4 g in 100 ml MilliQ H2O, (Fisher Scientific Co., catalog number M-13, Lot number 794098, EXP DEC 99).
    g) Ferrous sulfate heptahydrate, 0.06 g in 100 ml MilliQ H2O, (Fisher Scientific Co., catalog number I-146, Lot 712502, EXP DEC 99).
    h) Dissolve 16 grams of Difco nutrient broth powder into 1800 ml of Milli-Q H2O. Add 225 ml to each of 8 2-liter screw- capped Erlenmyer flasks. Autoclave 15 to 30 min at 121OC and cool to room temperature.
    SOP-UIB-BI-3 Παγε 3 οφ 6
    i) Dissolve individually the amounts of the compounds listed in b-d above in Milli-Q H2O, add 1 ml of e-g above to the solution, and bring to a total volume of 200 ml with MilliQ H2O. Filter sterilize through a 0.22- micron Nalgene filter unit. To each of the eight flasks containing 225 ml of cooled, sterile nutrient broth add 25 ml of the filtered salts-dextrose solution.

    5. Tryptic soy agar, (Difco Laboratories, catalog numnber 0369- 17-6, lot 58056JB, EXP OCT 99). Add 40 g to 1 liter of Milli-Q H2O, sterilize by autoclaving at 121OC for about 30 min, cool to about 50O C, and dispense O into petri dishes, approximately 20 ml per plate. Store at 2-8 C.

    6. Sheep blood agar, with 5% defibrinated sheep blood and tryptose blood agar base (Difco Laboratories, catalog number 0232-01-9, lot 10278JC, EXP OCT 99). Add 33 grams of tryptose blood agar base toO 1 liter of Milli-Q H2O. Sterilize by autoclaving at 121 C for about 30 min. After cooling to about 50-55OC, add the 5% (V/V) defibrinated sheep blood, mix, and dispense about 20 ml per petri dish. Store at 2-8OC.

    7. Phenol (GIBCO-BRL Life Technologies, P.O. Box 6009, Gaithersburg MD, 20897-8406, 301/840-8000, catalog number 15509- 011).

    8. Renografin-76 (Squibb Diagnostics, P.O. Box 5250, Princeton, NJ 08543, 800/631-5244, NDC 0003-0821-55, catalog number 82155, Lot 4A96314, EXP 1 JAN 99). This will be diluted to a concentration of 58% Renografin-76 in 1% phenolized water.

    Methods:

    O The spores are stored in the USAMRIID B-3 cold room, at 2- 8 C. The record of the cold room temperature made on on DIckson TempTrace recording device (MMCN E2797) will be kept in the record of this GRP study.) They were prepared as follows:

    1. With an inocuolating loop, a portion of Ames spores from a frozen tube was scraped off and inoculated onto sheep blood agar (SBA). The tube was returned to the -70OC
    freezer and the SBA culture was incubated overnight (about 16-18 hours) at 37OC in an atmosphere of 5% CO2.

    2. With an inoculating loop, growth from 2-5 isolated colonies was resuspended in about 10 ml of PBS. (Such a suspension is slightly turbid, and holds approximately 1 X 107 CFU/ml. The A540
    SOP-UIB-BI-3 Παγε 4 οφ 6 of the suspension is about 0.1, measured on a Bausch and Lomb
    model Spectronic 21 spectrophotometer, MMCN C0025.

    3. Approximately 50 to 100 μl of the suspension was inoculated into each of the 2-liter flasks containing Leighton-Doi medium. The screw caps of the flasks were loosened slightly (by about 1/2 turn) to allow for air exchange. The caps were held in place by a small piece of tape attached to both the flask and the cap. A large paper towel was taped over the cap and top of the flask to prevent aerosol formation during growth.

    4. The inoculated cultures in the flasks were placed into a New Brunswick controlled environmental incubator shaker, MMCN 04008, and incubated at 34OC to 37OC with reciprocating shaking (100 strokes per minutes) for about 24 h. The heating element was then turned off, and the cultures were incubated 3 to 4 more days at 25OC to 30OC.

    5. Culture samples from each of the flasks were examined at 1000X under phase microscopy to determine amount of sporulation and culture purity. Cultures visually judged contaminated with other microorganisms were autoclaved and discarded. Cultures with less than 80% sporulation were incubated another day under the above conditions.

    6. Uncontaminated cultures with greater than 80% sporulation were centrifuged to pellet cells in 500-ml polypropylene bottles, about 333 ml per bottle, in a GS-3 rotor, at 8,000 rpm, 20 min, 2-8OC, in a Dupont Sorvall RC-5B centrifuge, MMCN D7689.

    7. The spores pelleted tightly at the bottom of each bottle, and cells packed loosely on the top of the spores. The supernatant was removed with a 25-ml pipette and transferred to another flask for autoclaving and discarding. Before the final 25 ml was removed, it was swirled gently in the flasks, resuspending the cells but not many spores, from the bottom of the centrifuge bottle. This final 25 ml of supernatant and vegetative cells was transferred to the discard flask.

    8. After removal of the supernatant, 25 ml of sterile water for injection was added to the pellet in each centrifuge bottle. The pellet was resuspended by pipetting back and forth. The resuspended pellets were combined and dispensed into two 500-ml polypropylene bottles. After centrifuging as above, the supernatants were removed as above.

    9. Each pellet was resuspended in 125 ml of sterile water for
    SOP-UIB-BI-3 Παγε 5 οφ 6
    injection and centrifuged for 15 min at 8,000 rpm. After the supernatants were removed as above, each pellet was resuspended in 25-50 ml of sterile water for injection + 1% (w/v) phenol. The pellets were combined andO stored in sterile Polycarbonate flasks in the B-3 cold room (2-8 C).

    10. Spores from 8 separate runs were combined for purification on Renografin-76.

    11. The different bactches of spores in the 1% phenol were resuspended by swirling in the flasks, and then combined into a 500-ml polycarbonate flask.

    12. Renografin-76 (58%, v/v, in 1% phenol) was pipetted into four 250-ml polypropylene centrifuge bottles, 50-100 ml per bottle.

    13. The suspension of combined spore preparations was dispensed evenly by pipette onto the 58% Renografin-76. The bottles were then centrifuged for 1-3 hours at 2-8OC, HS-4 rotor, 6,500-7,000 rpm, in a Dupont Sorvall RC-5B centrifuge. At the end of the centrifugation, spores had pelleted at the bottom of the centrifuge bottles. Cells and other debris remained at the interface between the Renografin-76 and aqueous layers.

    14. The aqueous layer, interface and Renografin-76 layer were removed from the bottles by pipette, leaving the spores pelleted at the bottom of the bottles.

    15. To each bottle was added 25 ml of 1% phenol, and the pellets were suspended by pipetting.

    16. As above, the spore suspensions were dispensed onto 58% Renografin-76 (2 bottles) and centrifuged for a second time as above to pellet the spores. The aqueous layer, interface, and Renografin-76 layer were removed as above, and the spore pellets in the two bottles were resuspended in 1% phenol, 50 ml per bottle.

    17. The spores were pelleted by centrifugation as above for 15 minutes. The supernatants were removed by pipetting, and the spores were resuspended in 1% phenol, 50 ml per bottle.

    18. The spores were again pelleted by centrifugation and the supernatants were removed by pipetting. The spore pellets were resuspended in 1% phenol by pipetting, for aO total volume of about 125 ml. The spores were stored at 2-8 C in the B-3 cold room.
    SOP-UIB-BI-3 Παγε 6 οφ 6
    ————————————————————-

    I. Bruce Ivins, Bacteriology Division, 1 day prior to aerosol spore challenge will dilute the spores with sterile water for injection in a sterile GIBCO glass bottle to approximately 1.5 X 109 CFU/ml, with a total volume in ml determined by the formula: (# Monkeys to be challenged + 1) X (7.5 ml). The diluted spore suspension will be placed on ice in an ice bucket in the B-3 cold room.

    II. Bruce Ivins, Bacteriology Division, on the day of aerosol spore challenge will
    a) Heat theO diluted spore suspension for 45 min at approximately 60 C in a water bath in B313 (no MMCN, Blue M Electric Company, Model SPB-1070, calibrated in DEC 94 by USAMRIID Medical maintenance personnel).
    b) Dispense the spores into screw-cap polypropylene tubes, about 7.0 – 7.4 ml per tube and seal each tube with vinyl tape, saving a sample of the spores (at least 0.1 ml) for plate count. The number of tubes of spores should be one more than the number of monkeys to be challenged.

    2 c) Dilute the above saved spore sample to approximately 3 X 10 CFU/ml with sterile Milli-Q water, and plate out 0.1 ml onto each of 5 TSA plates. The plates will be incubated in B304 at approximately 37OC for 16 to 24 h in a Lunaire model B10665G incubator, calibrated by USAMRIID Medical Maintenance in DEC 94, and then colonies will be counted by persons able to visualize, differentiate and count individual B. anthracis colonies on TSA. The concentration of spores in the tubes for aerosol challenge will be determined.
    d) Double bag the tubes of spores and place in the B-3 passbox.
    e) Have an individual put the double bagged spores into a plastic bag in a metal box, and take the box to the 1412 airlock and remove the top of the box so that personnel in the 1412 “hot suite” can remove the spores for aerosol challenge.
    Prepared by:____________________________________________________
    Bruce E. Ivins, Ph. D., USAMRIID Bacteriology Division
    SOP-UIB-BI-3 Παγε 7 οφ 6 Date:__________________________________________
    Study Director:_ _____________________________
    USAMRIID Toxinology Division Date:__________________________________________

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