Colleague Rebuts Idea That Suspect’s Lab Made Anthrax in Attacks/ NY Times

The New York Times’ Scott Shane reports on the testimony of Henry E. Heine, former Ivins colleague, at the National Academy of Sciences panel reviewing the FBI’s science at its meeting today.

… Asked by reporters after his testimony whether he believed that there was any chance that Dr. Ivins, who committed suicide in 2008, had carried out the attacks, the microbiologist, Henry S. Heine, replied, “Absolutely not.” At the Army’s biodefense laboratory in Maryland, where Dr. Ivins and Dr. Heine worked, he said, “among the senior scientists, no one believes it.”

… He told the panel that biological containment measures where Dr. Ivins worked were inadequate to prevent the spores from floating out of the laboratory into animal cages and offices. “You’d have had dead animals or dead people,” he said…

More thoughts of Dr. Heine were reported in the Frederick News-Post.

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Ed Lake
Ed Lake
12 years ago

Science Magazine has an article here: http://news.sciencemag.org/scienceinsider/2010/04/ex-usamriid-scientist-defends-br.html

It says Dr. Heine has NO EXPERIENCE making anthrax spores.

They asked Dr. Adam Driks at Loyola, who makes spores routinely, how long it would take to make the spores that where in the envelopes. Driks says "a number of days."

Heine claimed it would take a year.

It's nice when knowledgeable experts state facts which dispute the opinions of people who do not know what they are talking about.

Meryl Nass, M.D.
Meryl Nass, M.D.
12 years ago

How long does it take to grow and dry 10-20 trillion anthrax spores?

That depends on the size of the fermentation vessels and the machine used for drying.

With the right equipment it would take days. With what his colleagues say was available to Bruce Ivins in the containment lab at Fort Detrick, it would take considerably longer.

Ellen Byrne
Ellen Byrne
12 years ago

Thanks, Dr. Nass!

It's nice when knowledgeable experts state facts which dispute the opinions of people who do not know what they are talking about.

Ed Lake
Ed Lake
12 years ago

Meryl,

How long would it take Ivins to create the media spores if he simply opened up the autoclave in his lab and pulled out the growth plates that were always accumulating in there for weeks?

I suspect that Ivins' colleagues would merely say "that is against procedures," therefore he'd never do that.

Also, you do not need a "machine" for drying. More and more scientists are saying that Ivins probably used chemicals to dry the Senate spores, because that drying procedure would produce spores that do not tend to stick together.

According to one scientist the FBI interviewed, Ivins learned how to dry spores that way when he visited Dugway. If he didn't learn it there, he could have learned it off the Internet.

Ed Lake
Ed Lake
12 years ago

Meryl asked, "How long does it take to grow and dry 10-20 trillion anthrax spores?"

There was less than 1 trillion spores in each of the 2 Senate letters.

The media letters were reportedly about 90 percent debris. That means that all five media letters probably had less than a trillion spores in total.

So, we're talking about less than 1 trillion spores for the first mailing and about 2 trillion spores for the second mailing. 10-20 trillion anthrax spores is just more nonsense from people who desperately want to believe that Ivins couldn't have done it. Because if Ivins DID do it, that means they failed to notice it.

Meryl Nass, M.D.
Meryl Nass, M.D.
12 years ago

The recovered letters had all been through sorting machines. The plume of spores above the sorting machine allegedly reached over 20 feet high. The pores in the paper were much larger than the size of the spores. Thus there were many fewer spores when the letters were found than when they were sent. Bill Patrick suggested that 2 grams could be sent in a letter without being noticed. I think that amount is a reasonable estimate of how much material was in each envelope, especially since there was an underwhelming response to the first set of letters. So the perp(s) would have wanted to send something much more powerful in the second round.

How many letters were there in all? Only 4 were recovered. The letter to AMI was not recovered. People connected to ABC, NBC and CBS developed anthrax, but only a letter to NBC was recovered. At a minimum, 3 are missing.

I think an estimate of 10 total letters is reasonable.

Whichever way you slice it, trillions of spores were used, since everyone agrees there were about a trillion spores each left in the Daschle and Leahy letters.

The early letters had lots of debris, while the second set of letters had virtually no debris and were uniform in size. How many spores were lost during processing, to achieve such high purity and uniformity? I don't know.

When growing anthrax to make vaccine, the growth process takes several days, and the yields vary enormously between batches. Growing in the blood of a live animal, I think anthrax can reach one billion vegetative organisms max per gram of blood, if memory serves. I am guessing you might get a couple/few billion max per agar plate.

So we may be in the range of 1-10 thousand agar plates (or more) needed to grow enough for the letters, assuming little loss to processing.

However, I have never worked in a biodefense lab, so my back-of-the-envelope calculations may be wrong.

Andy
Andy
12 years ago

Dear Ed,
Heine is probably right!
If you look at the labbook data released by the authorities:
Daschle letter (SPS02.57.03) contained 2.1 x 10e12 cfu/g (that's viable spores only).
Assuming 1.5 g spores were in this single (!) letter, at least 3.15 x 10e12 spores had to be produced.

Now have a look at the RMR1030 Ames data:
13 batches of spores were prepared by Ivins and a technician between 11/20/95 and 11/18/96. (This includes the 2 liter production runs and renocal purification).
This process yielded 1.1 x 10e8 spores/ml original culture and 3 x 10e12 spores total.

This shows, that one (1!) year of work yielded the amount of spores present in the Daschle letter.

Then the new task was to prepare the infamous RMR1030 containing 10e13 spores total. This would have taken 65 and 130 weeks at 1 and 2 production runs per week respectively. -> Dugway was contracted.
10e13 should be about the total amount of spores sent around in the letters.

Judging from his papers, the so-called expert (Driks) has never prepared concentrated spore suspensions and of course no dry powder. He's speaking from the normal microbiology lab experience. At first, I thought too "What's the problem – Just inoculate some medium, put it the incubator and there you go…"
Turns out, it's not as easy.

Regarding the Powder: There is an analysis of 7 different powder preparations in the evidence. It's all crap and far, far away from the letters. Partially no viable spores at all, or the majority of spores is not viable and clumpy…

—-
Regarding the acetone. I don't know whether you've tried this, but when you add acetone to petridishes the plastic get sticky and dissolved

Andy
Andy
12 years ago

@Ed
Regarding you petri dish hypothesis. Here we are talking about around 1e8 cells per plate. I don't know whether you have ever tried to scratch Bacillus from "not fresh" (sporeforming) culture. Doesn't work very well and you most likely recover < 1e7 or 1e6 spores per plate. (Actually, I had really problems to get a decent amount of Bacillus off the plate when it was laying around in the fridge for a week).

Anonymous
Anonymous
12 years ago

I notice Ed Lake is back again spouting out his usual nonsensical garbage. Lake's latest fabricated statement is:
"Also, you do not need a "machine" for drying. More and more scientists are saying that Ivins probably used chemicals to dry the Senate spores, because that drying procedure would produce spores that do not tend to stick together."

For the record – when Lake mentions "chemicals" used for drying he his referring to azeotropic drying. He is, as usual, completely incorrect that this technique does not involve a "machine". It involves a highly specialzed distillation apparatus – and furthermore the exact ratios of "Chemical" (in the case of spores acetone or ethyl acetate could possibly be used) to water slurry is critical – as is the distillation temperature and pressure conditions. In other words – impossible for Bruce Ivins to have clandestinely set up inside Detrick even if he had the know-how (which he didn't). The fact that he would still have produced a dry powder aerosol which would have completely contaminated the lab is another little problem. But, hey, when Lake is involved who cares about facts? For some unknown reason Lake is on an obsessive, almost religious, mission to prove to the world that Bruce Ivins is a homicidal maniac. His fantasies that Bruce coerced a six year old boy from his wife's day-care into writing the anthrax letters and envelopes are the product of an especially fertile (some might say disturbed) imagination.

References:

http://pdfserve.informaworld.com/954243__790515467.pdf
The culture was incubated at 30◦C in a 10 L fermentation
vessel with an agitation rate of 250 RPM and an aeration rate
greater than 0.5 volumes min−1. Sporulation was generally complete
within 24 h. Spores were collected by simple centrifugation
to remove spent media. The pelleted material was dried by
a proprietary azeotropic method.

http://en.wikipedia.org/wiki/Azeotrope

http://www.agriculture.purdue.edu/fnr/faculty/Eckelman/documents/d1971256.pdf

Anonymous
Anonymous
12 years ago

FrederickNewsPost.com has the full text of the FBI documents ***in Google-searchable form***. (For example, the text you forwarded can be fond on page 2 of the PDF at http://www.fredericknewspost.com/media/pdfs/ivins_investigation_13.PDF.)

Google searches of the form "fermentor site:www.fredericknewspost.com" and "fermenter site:www.fredericknewspost.com" yield the following fermentor/fermentoe-relevant hits:

a fermenter for growing labs where Ba work primarily occurred were rooms[ however, …. J to allow the moving of a fermenter too large to fit …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_19.PDF

File Format: PDF/Adobe Acrobat – View as HTML
IVINS' Knowledge of the Use of Fermenters to Grow Anthrax … fermenter-grown. IVINS based this judgment on his observation that …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_2.PDF

the fermenter was very messy due to the parts and supplies required for operation. … in the fermenter, but speculated that recombinant BA preps made …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_11.PDF

five liter capacity fermenter can now produce approximately five grams of protective antigen per liter. (U) EC. Serial 109) re …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_16.PDF

one fermentor as having been in … Additional fermentors of interest have been identified …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_13.PDF
[THIS IS THE SECTION YOU FORWARDDED…WHICH REFERS TO FERMENTERS RANGING FROM 150 L TO 5 L, INCLUDING A 5 L FERMNENTER ASSIGNED TO IVINS.]

physical location for this fermentor but was sure it was not located in … logic behind purchasing the fermentor. IVINS also relayed to that he recalled …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_29.PDF
[THIS SECTION DISCUSSES THE 5 L FERMENTOR THAT HAD BEEN ASSIGNED TO IVINS AND THAT WEAS "LOCATED WITHIN THE HOT SUITE."]

was a fermentor in Suite B4 used for protective antigen (PA) and lethalb6 factor studies. The fermentor was in a laboratory considered to be k>7c …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_27.PDF

There has never been a fermentor Building 1412 and … too dangerous to have grown Ba Ames in a fermentor. was m. Building| | while was at USAMRIID. …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_16.PDF

spores to inoculate a fermentor. IVINS continued to review the … germinating medium used when inoculating a fermentor. IVINS …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_2.PDF

Copies of hand receipts showing IVINS had a New. Brunswick, BioFlo III fermentor and a lyophilizer issued to him. ,iMS. Page 2. Page 3 …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_4.PDF

originally obtained these fermentors around 1980-1985. [. The 100-liter fermentorslT … |"Big Bertha" is the 100-liter fermentor identified as stock …
http://www.fredericknewspost.com/media/pdfs/ivins_investigation_11.PDF
[THIS SECTION REFERS TO FIVE FERMENTERS: 100 L, 30 L, 20 L, and 3 L.]

Ed Lake
Ed Lake
12 years ago

Dr. Nass wrote: "everyone agrees there were about a trillion spores each left in the Daschle and Leahy letters."

In reality, the only actual data we have comes from the New York Times which stated that the powder in the Leahy letter weighed .871 grams. That's equivalent to 871 billion spores.

"The pores in the paper were much larger than the size of the spores.'

In reality, the pores in the paper are not open holes like in a sieve. The paper is a mesh of paper fibers, and the spores have to work their way through the mesh. The thickness of a sheet of paper is about 40 times the diameter of a spore. So, spores will bunch up in the fibers and prevent the escape of the spores behind them.

Also, the spores were wrapped inside a letter folded with the pharmaceutical fold, which means they had to get through at least TWO sheets of paper to reach the outside of the envelope.

I seriously doubt that more than 5 percent of the spores escaped the letters.

"How many spores were lost during processing, to achieve such high purity and uniformity? I don't know."

Perhaps some data from Professor Richard Ebright will help. Here's what he told me back in February of 2003:

"For a broad range of bacterial species, ~1 liter of culture yields ~1 gram
of cells. With a sporulation efficiency of, say, 70% and a purification
efficiency of, say, 70%, ~2 liters of culture would yield ~1 gram of spores."

Ed Lake
Ed Lake
12 years ago

Anonymous wrote: "For some unknown reason Lake is on an obsessive, almost religious, mission to prove to the world that Bruce Ivins is a homicidal maniac.

I simply explain the FBI's case, so I'm attacked for being on a "religious, mission to prove to the world that Bruce Ivins is a homicidal maniac." That's a True Believer's way of avoiding discussing facts.

"His fantasies that Bruce coerced a six year old boy from his wife's day-care into writing the anthrax letters and envelopes are the product of an especially fertile (some might say disturbed) imagination."

And rather than look at the facts about the handwriting, I'm attacked as being "disturbed" because Anonymous refuses to discuss any facts which do not conform to his beliefs.

Dr. Nass, is that also your idea of discussing issues – personally attacking those who do not agree with you?

Meryl Nass, M.D.
Meryl Nass, M.D.
12 years ago

I should have said, I believe we agree that the letters contained about 1 gram of spores.

Although you are right about the paper and envelopes, a sizable amount of spores left the envelopes. Let's agree we have no way to determine how much anthrax the letters initially contained. Your suggestion of 5% lost is simply a guess.

Bob Ebright was making up numbers. They may be correct. But they were not specific to the methods of growth and purification of the letter spores (since we don't know what those entailed) and because anthrax growth is extraordinarily variable (for example, 2 orders of magnitude different concentration in lots prepared the same way) these estimates are also guesses.

Meryl

Anonymous
Anonymous
12 years ago

Ebright apparently wrote:

"For a broad range of bacterial species, ~1 liter of culture yields ~1 gram
of cells. With a sporulation efficiency of, say, 70% and a purification
efficiency of, say, 70%, ~2 liters of culture would yield ~1 gram of spores."

This is practically useless information.
Of course, Lake prefers not to look at the FACTS, but rely instead on some misguided generalization made by someone who has never before made spores in his life.

The FACTS about the yields from Detrick and Dugway's best known methods for anthrax spore production can be found in several places. This includes Dr Clare Fraser-Liggit's presentation to the National Academy of Science's FBI anthrax science audit.

The single flask of RMR-1029 consisted at its origination date of 30g of Ames anthrax spores in a slurry of 1 liter of water. The resources needed to make this 30g of spores consisted of a combination of 12 x 10 liter fermentor runs at Dugway Proving Ground and 22 flask culture lots made at USAMRIID. Dr Bruce Ivins had calculated that to make 30g of spores at USAMRIID it would take approximately one year of work, which is why USAMRIID contracted the large fermentor runs at Dugway in order to fulfill their need for spores for animal vaccine challenge studies.
This is documented on slide 31 here:
http://caseclosedbylewweinstein.files.wordpress.com/2010/01/fraser-liggett-et-al-the-genomics-behind-the-amerithrax-investigation.pdf

Thus it took 165 liters of runs to make 30g of spores.
That's a yield of 1 gram of spores per 5.5 liter production run. Thus at least one full production run in Dugway's fermentor would be needed to make the spores for just one envelope. One production run would produce 1.8g of spores from 10 liters. After drying, transferring to the envelope and extracting from the envelope accounting for losses all along the way (this stuff floats into the air when it's handled) one might eventually weigh 0.87 grams – the amount extracted from the Daschle envelope.

The above FACTUAL documentation of how RMR-1029 was made is exactly in line with Dr Heiene's estimates presented to NAS this week.

Lake will of course state this is all wrong – even although it is the FACTUAL account of authorized record. Lake will fantasize that Bruce Ivins must have known some secret method of making spores.
Maybe that's why Detrick spent $100,000 contracting Dugway to make spores – they like wasting government money when they all along knew a secret method of making enough spores in 3 days to keep their animal challenge vaccine program going for years 😉

Anonymous
Anonymous
12 years ago

Lake wrote:

"I simply explain the FBI's case, so I'm attacked for being on a "religious, mission to prove to the world that Bruce Ivins is a homicidal maniac." That's a True Believer's way of avoiding discussing facts."

Lake is the one avoiding discussion of the facts. When finding out that the "chemical" used to dry spores is, in reality, part of a complex piece of equipment after claiming it wasn't he simply, as usual, resorts to name calling. He now refuses to admit that, once again, he has been shown to spread false propaganda on the internet at every possible opportunity.

Stating that most reasonable people would believe Lake has an over fertile and potentially disturbed imagination for invoking children from imaginary day care centers into his theories about Bruce Ivins is hardly a personal attack – it's just a statement of fact.

Ed Lake
Ed Lake
12 years ago

A scientist with considerable experience making spores just sent me an article from 1984 titled “Single, Chemically Defined Sporulation Medium for Bacillus subtilis: Growth, Sporulation, and Extracellular Protease Production.”

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC214739/?page=1

It’s an article about Bacillus subtilis, but the principles can be applied to Bacillus anthracis.

The scientist explained to me that the report shows that they grew spores in a 300 milliliter flask.

They diluted the spore material in the flask 100 times to do the spore counts. They counted 1 to 2 billion spores in the diluted sample. That equates to 300 to 600 billion spores in the 300 milliliter flask.

The scientist confirms that that means that you would get 1 trillion to 2 trillion spores in a 1 liter flask.

And he confirmed that would be about 1 gram of dried spores.

Any arguments?

Meryl Nass, M.D.
Meryl Nass, M.D.
12 years ago

No argument about max growth of 1-2 x 10 to the 9th power of vegetative cells/ml. There is some loss as these cells become spores. There is additional loss as they are dried and then processed. We don't know how much loss those processes entailed.

I am going to end this thread now.

Meryl

Anonymous
Anonymous
12 years ago

"Any arguments?"

Yes. Your scientist is clearly confused.

The “scientist” who sent you this apparently got lost in wishful thinking and forgot to read the abstract:

“The composition and application of a single, chemically defined medium or growth and sporulation of Bacillus subtilis is described. At 37 degrees C cells grew with a doubling time of about 40 min; cultures attained near-maximal spore formation (70 to 80% by 12 h after the end of exponential growth and produced 1 X 10(9) to 2 X 10(9) heat-resistant free spores at 24 h. Dipicolinic acid production was completed between 7 and 11 h. Cells grown in the single, chemically defined medium excreted levels of serine and neutral proteases comparable to those excreted in nutrient broth medium.”

A 300ml flask was used, and 50ml of CDSM media was used. Thus 50ml of media yielded 10^9 spores. That means 50 liters of media would be needed to yield 1 gram (10^12 spores). In other words, with this method even 50 liters would not yield enough to fill even one envelope.

The Dugway fermentor anthrax was capable of yielding 1 gram of Ames anthrax spores with only 5.5 liters.

The fantasies of your anonymous “scientist” contact are off by more than a factor of one thousand. Try again.

Meryl Nass, M.D.
Meryl Nass, M.D.
12 years ago

I wanted to close this thread but received reasonable posts from Andy and Ed Lake afterwards, so will summarize them. Ed says a 1984 paper's Figure 1

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC214739/?page=2

gives Bacillus subtilis yields using different media. Yields ranged from 2 x 10 to the 8th spores/ml to 2 x 10 to the 9th spores per ml.

Andy notes that Leighton Doi, the usual USAMRIID medium used for anthrax growth,yielded 1 x 10 to the 8th spores per ml.

What we do not know is whether the growth curves for B. subtilis have any bearing on the growth of B. anthracis used in the letters.

Even if the perp(s) managed to generate 10 to the 9th spores per ml, approximately 10 liters of medium would be needed to fill the letters.

What I want to know is where is the Bacillus contaminant (present in the first set of letters) to be found? Somehow it contaminated the Ames anthrax, and therefore traces of it should remain in the area where the anthrax was processed. If it could not be found in the lab where Ivins grew his anthrax, then the anthrax for the first letters was not grown in that lab.

Meryl

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