* Who was the colleague with whom Dr. Heine says he did research regarding antifoam in creating aerosols?
Posted by DXer on April 25, 2010
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CASE CLOSED ... what really happened in the 2001 anthrax attacks? 
DXer said
USAMRIID has (presumably inadvertently) not uploaded Batch 29 which 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 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. I have cloned the USAMRIID and FBI computers in case they misplace any documents. ; )
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:
DXer said
3/31/2005 –
Because IVINS estimated it would have taken he and _____________________ approximately one to two years to produce 1,000 milliliters of concentrated spores using their standard flask fermentation method, USAMRIID contracted with Dugway Proving Ground which agreed to produce the spores in batches at Dugway Proving Ground using fermenters and then ship the batches as they were completed to USAMRIID for purification and use.
DXer said
By email dated October 11, 2004, Dr. Ivins wrote:
“If centrifugation through hypaque causes the foaming problem, but the spores still need to be cleaned up beyond water washes, perhaps centrifugation through sucrose might be a suitable replacement (for the Hypaque). ____ if washing doesn’t work, then we’ll try an experiment here with spores from the same prep, but treated as follows:
a) Washed with water only
b) Washed with water and centrifuged through Hypaque gradient
c) Washed with water and centrifuged through Sucrose gradient
______ could we slip the above three preps in sometime for a spray factor determination if necessary?
I remember seeing the ____ lot 15 (or was it 16 ___ that had been hypaque purified, and i didn’t foa at all. The _______ material was made in a fermentor containing antifoam. I’m wondering if this is the difference.
Since Hypaque is fairly soluble (89g/100 ml of water at 20C), I’m surprised that washing in water wouldn’t solubilize the material away from the spores. One easy way to see if Hypaque is the problem would be to take some purified spores that don’t foam, add some Hypaque to them, then spray them to see if the spray factor changes.”
A said
Hi,
maybe there is a misunderstanding. In the final product there should not be any hypaque. You just centrifuge your spore preparation through a hypaque gradient to separate (->purify) spores by their density. Following Spores have to be recovered from the gradient and washed with water to remove hypaque residues
BugMaster said
If a sucrose gradient (or other application utilizing sucrose) was used to prepare a portion of the attack material, it could account for the trace amounts of tin detected (and contribute somewhat to the silica content).
Limestone is used in the processing of sugar cane, and in many regions where sugar is produced, contains quite a bit of tin.
DXer said
FBI’s anthrax probe explores possibility tha
– Dec 2, 2001
“They didn’t talk to me about my personal experience,” said Friedlander, a physician and leading anthraxexpert. “They asked me about other personnel.”
I think Dr. Friedlander is above suspicion from start to finish. And I think that of all the personnel at USAMRIID with whom I know anything about — especially Dr. Ivins now that I’ve read many hundreds of his emails.
Bruce Ivins supplied virulent Ames to a scientist, Tarek Hamouda, who was the lifelong friend of a hero of mine, “Tawfik” Hamid. Dr. Hamid was recruited by Ayman Zawahiri in medical school but rejected the indoctrination when he was asked to help bury a security officer near the mosque..
Given that Tarek had gone to Cairo Medical School at the heyday of Sadat’s assassination and Ayman Zawahiri’s and Ali Mohammed’s recruitment at the Medical School, it would have been dubious to invite him to work alongside Bruce Ivins without proper vetting. In light of Bin Laden’s declaration of war and Zawahiri’s longstanding recruitment attempts aimed at infiltration US biodefense, what vetting was done?
Dr. Hamouda graduated medical school in December 1982 and then got his PhD in microbiology department (where Ayman’s sister Heba taught) in 1994. He was in charge of the DARPA anti-infective project a few years later.
The early 1980s — when TH got his MD — was when Ali Mohammed recruited Dahab at the medical school. Dahab moved to join him in the Silicon Valley and who he trained to make lethal letters.
Dahab and Ali Mohammed travelled to Afghanistan and told Bin Laden that they had recruited 10 sleepers in the US. Who were the sleepers?
What vetting was done of Dr. Hamouda? Dr. Ivins did not even know that the scientist arriving was not a US citizen.
Dr. Hamouda thanked Dr. Friedlander and Dr. Patricia Fellows (Former Colleague #2 in the documents) for providing technical assistance. What technical assistance did they provide?
Was AF, a top anthrax expert for the Army, the one experimenting with an antifoam in creating aerosols with Dr. Heine?
Dr. Hamouda’s decontamination agent was tested at the US Capitol and the company pitched hand cream to postal workers.
The company received $80 million in in investment — $50 million from Perseus, which was headed by Richard Holbrooke, now #3 at Department of State in charge of Pakistan and Afghanistan.
It’s time for some basic questions to be answered.
First, who did Dr. Heine do the experiments involving antifoam in creating aerosols with?
Second, did the FBI’s anthrax expert, John Ezzell, use an antifoaming agent or a silanizing agent in the slurry before lyophilization?
The DARPA researchers were experimenting with the effect of sonication and Corona Plasma Discharge on Ames spores.