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FBI Ignored, Hid Data Potentially Excluding Bruce Ivins as Anthrax Killer

9:16 am in Uncategorized by Jim White

FBI photos of the material in the letter sent to Senator Pat Leahy (left) and to the New York Post (right), from the report.

A report from McClatchy provides important new evidence and analysis in the FBI’s Amerithrax investigation of the 2001 anthrax attacks. The report shows that the FBI ignored as potentially erroneous a measurement of silicon in one anthrax sample and then hid this information from Congressman Jerrold Nadler (D-NY). Even more importantly, the high silicon measurements in at least two samples also were coupled with high tin measurements, opening up the possibility that silicon was added to the attack material in a form that is not mentioned in any of the FBI documents. Significantly, it is virtually impossible that Bruce Ivins, whom the FBI has concluded acted on his own to carry out the attacks, would have been able to perform the necessary chemical manipulations involved in this treatment of the spores.  Ivins likely also would not have had access to the necessary laboratory equipment to perform this treatment.

The presence of silicon and how it may have gotten into the anthrax material has been a point of great controversy throughout the entire investigation. This question is important because the chemical nature of the silicon and the level at which it is present is presumed to be an indicator of whether the anthrax spores have been “weaponized” to make them suspend more readily in air so that they are more effective in getting into the small passageways of the lungs of the intended targets of the attack. Early in the investigation, Brian Ross published “leaked” information that the spores had been weaponized through addition of bentonite and that Iraq had a weaponization program that used bentonite. This report turned out to be false, as no evidence for bentonite has been found. A more sophisticated type of weaponizing would rely on mixing the spores with nanoparticles of silica (silica is the common name for the compound silicon dioxide) to make them disperse more easily.

The FBI carried out a special form electron microscopy that could identify the location of the silicon in the spores from the attack material. They found that the silicon was in a structure called the the spore coat, which is inside the most outer covering of the spore called the exosporium. If silica nanoparticles had been used to disperse the spores, these would have been found on the outside of the exosporuim (see this diary for a discussion of this point and quotes from the scientific literature) because they are too large to penetrate it.  No silicon signature was seen on the outside edge of the exosporium.  What is significant about the type of silicon treatment suggested in the McClatchy piece is that both high silicon and high tin measurements were found in several samples and that there is an alternative silicon treatment that would involve a tin-catalyzed polymerization of silicon-containing precursor molecules. McClatchy interviewed scientists who work with this process and they confirmed that the ratio of silicon to tin found by the FBI is in the range one would expect if such a polymerization process had been used. Read the rest of this entry →

McClatchy Points Out Key FBI Failure in Amerithrax Investigation

5:58 am in Uncategorized by Jim White

The infamous RMR-1029 flask. Where is the B. subtilis flask?

In an article published Wednesday evening on their website, McClatchy points out yet another failing in the FBI’s Amerithrax investigation of the 2001 anthrax attacks that killed five people. The article focuses on the fact that the FBI was able to get a clear genetic fingerprint of a bacterial contaminant that was found in the attack material mailed to the New York Post and to Tom Brokaw (but not to either Senator Daschle or Senator Leahy). This contaminant, Bacillus subtilis, is used in some cases by weapons laboratories as an anthrax simulant, because its behavior in culture and in drying the spores is very similar to Bacillus anthracis but it is easier to handle because it is not pathogenic.  I covered the FBI’s failure to link this B. subtilis contaminant to Ivins in this diary in February of 2010.

A key finding in the McClatchy article comes from an FBI memo they obtained:

One senior FBI official wrote in March 2007, in a recently declassified memo, that the potential clue “may be the most resolving signature found in the evidence to date.”

The genetic fingerprint of the B. subtilis contaminant, in other words, was a very definitive clue that needed to be traced to the culprit. The problem, as McClatchy points out, is that the FBI never got a match to materials from Ivins or from anyone else:

Yet once FBI agents concluded that the likely culprit was Bruce Ivins — a mentally troubled, but widely regarded Army microbiologist — they stopped looking for the contaminant, after testing only a few work spaces of the scores of researchers using the anthrax strain found in the letters. They quit searching, despite finding no traces of the substance in hundreds of environmental samples from Ivins’ lab, office, car and home.

LSU anthrax researcher Martin Hugh-Jones makes the key point in his discussion with McClatchy, in response to being informed the government tested thousands of samples for a match to the B. subtilis contaminant, “But were they thousands of the right samples?”

The significance of B. subtilis being present in some of the attack material cannot be overstated, especially when the FBI admits that they were able to obtain a unique DNA fingerprint of the strain that was present. As I wrote last year, I suspect that Defense Department personnel at either Dugway or Batelle cultured the material used in the attacks and noted personnel there have worked with both anthrax and B. sublitlis:

Even though Ivins can’t be linked to the particular B. subtilis strain used, there is a documented case of B. subtilis being used as a B. anthracis simulant at another facility where we already know that much of the material that went into RMR-1029 was produced. Recall from this diary that I analyzed the available information about the amount of B. anthracis used in the attacks and found it highly unlikely that Ivins could have cultured the large amount of spores used in the attacks with the equipment and time he had available. Much of the material in RMR-1029 was produced at Dugway.

On December 13, 2001, Judith Miller published an article in the New York Times, where she disclosed that “government officials have acknowledged that Army scientists in recent years have made anthrax in a powdered form that could be used as a weapon.” She further pointed out that this work occurred at Dugway in 1998. It should be noted that the anthrax produced at Dugway for Ivins that went into RMR-1029 was cultured in 1997.

The Miller article then goes on to quote scientist William C. Patrick on how he coached scientists at Dugway in drying a pound of highly purified anthrax spores in 1998. Miller quotes a Dugway spokesperson as saying a strain different from Ames (the parent of RMR-1029) was used in the drying experiments. Note that a pound of spores is enough dried spores to produce hundreds of letters with the one to two grams of dried spores thought to be in each letter. But in a further bit from the Dugway spokesperson, we have this:

She said Dugway did make one- pound quantities of Bacillus subtilis, a benign germ sometimes used to simulate anthrax.

We know that the key anthrax work at Dugway and Batelle was carried out by the Defense Intelligence Agency. I find it highly unlikely that this agency would be completely forthcoming with the FBI in sharing samples of all of the B. anthracis or B. subtilis strains in its collection, so Hugh-Jones was right to question whether the FBI tested the right samples for a match to the key B. subtilis contaminant. The fact that all of Ivins’ materials were available to the FBI, and that the FBI was able to carry out extensive environmental sampling (albeit several years after the preparation of the attack materials) where Ivins worked and lived comes very close to excluding Ivins as a suspect since the contaminant was not found in association with him.

Buried in the McClatchy article is an admission from a source close to the investigation that seems to shed some light on why the FBI continues to insist that Ivins was the sole attacker, even going so far as to concoct an after-the-fact “forensic psychiatric profile” in an effort to bolster their case:

“If they ever had any doubts, once he committed suicide, they had to unite,” this person said. “Otherwise, you’ve driven an innocent man to suicide. And that’s a terrible thing.”

Yes, driving a man to suicide is a terrible thing. But is it any less terrible to continue to insist on the guilt of a man who cannot conclusively be proven to have carried out these horrendous attacks ten years ago?

Would the NAS Report Have Led to Reasonable Doubt in an Ivins Trial?

12:02 pm in Uncategorized by Jim White

FBI photos of the material in the letter sent to Senator Pat Leahy (left) and to the New York Post (right), from the report.

It seems very likely to me that had Bruce Ivins not died, the analysis carried out by a panel from the National Academy of Sciences in assessing the scientific evidence tying Ivins to the 2001 anthrax attacks would have led to reasonable doubt on whether Ivins carried out the attacks. For this post, let us concentrate only on the NAS response to FBI claims on the spores used in the attack, especially with regard to how the spores were prepared.

What we do know from the report is that the spores used in the attacks did not come directly from Bruce Ivins’ RMR-1029 flask, but had to undergo a culturing step if RMR-1029 was even the source. Also, the spore material in different individual mailings differed in purity and particle size.  Silicon was present in the spores, but was not added as a step to “weaponize” the spores.  Instead, the silicon was incorporated into the coating of the spores themselves.   Finally, the NAS panel did not feel there was sufficient evidence to support the FBI claim that a highly skilled person had prepared the material.

Overall, the importance of the primary conclusion of the NAS report cannot be overstated (p. 4 of the report as marked, all references will use internal page numbers, not pdf numbers from my pre-publication copy):

It is not possible to reach a definitive conclusion about the origins of the B. anthracis in the mailings based on the available scientific evidence alone.

A good defense attorney probably would need no more than that conclusion to establish reasonable doubt in a trial.  But the details on how the panel reached that conclusion are important.

In this post, I discussed whether Ivins could have produced all of the material used in the attacks with the equipment he had available and without drawing attention to himself.  That post opened with a discussion of how many spores were known to have been present in the attacks.  I looked at what was in the FBI’s report, filled in some gaps with my experience in microbiology and came to the conclusion that somewhere between four and seventy liters of liquid culture could have produced the attack material.  The NAS report comes to a similar conclusion on page 62:

Thus, cultivation in the range of 2.8 to 53 liters of liquid medium would have been required to produce the spores required for the letters (see Table 4.2).

Interestingly, the panel also calculated that it would have required between 463 and 1250 agar plates if the attack material had been produced on solid medium.  It seems highly unlikely Ivins could have cultured this many extra Petri dishes without someone in the lab taking note and reporting it to the FBI once the investigation began.

The photo above shows how dramatically different the highly purified material in the letter sent to Senator Patrick Leahy’s office was when compared to the material mailed to the New York Post.

There had been much speculation early on in the press that the material sent to Senator Leahy’s office had been “weaponized” by the addition of materials including silicates to make tiny particles remain suspended in the air so that they could be inhaled.  The report documents that although silicon is found in the spores used in the attacks, the silicon is localized in the spore coats.  This point is driven home very clearly in this photo, where the silicon can be seen “lighting up” within the outer lining of the spores:

Many experiments were carried out in an attempt to match the silicon content, the particle size distribution and the degree to which the final material would remain suspended in air.  I will rely here on the entire summary the panel provided on the issue of silicon in the spores (page 71):

The substantial effort devoted to the characterization of silicon in Bacillus spore coats resulted in new fundamental insight into microbial processes and the development of new or enhanced analytical measurement technology. (Table 4.4 presents a summary of the analytical results.) Elemental analysis of the letter samples showed that 1) the silicon content was high, 2) most of the silicon was incorporated in the spore coat, 3) the majority of spores in the samples contained silicon in the coat, and 4) no silicon was detected in the form of a dispersant in the exosporium.

The bulk silicon content in the Leahy letter could be completely explained by the amount of silicon incorporated in the spores during growth. (Not enough material was available to make this comparison for the Daschle letter.) In contrast, the New York Post letter had significant bulk silicon content, far exceeding that contained in the spores.

No studies have considered the effect of the chemical form of silicon (e.g., silicate impurity versus polydimethylsiloxane antifoam agent) on uptake. The inability of laboratory experiments to reproduce the silicon characteristics of the letter samples is not surprising given the complexity of the uptake mechanism.

A few spores analyzed from RMR-1030 contained silicon in the coat, but none of the spores analyzed from RMR-1029 contained silicon in the coat. Therefore, the letter samples could not have been taken directly from the flasks—a separate growth preparation would have been required.

The material in the Daschle and Leahy letters was reported to have “a high level of purity” and to have electrostatic properties that caused it to disperse readily upon opening of the letters. These properties should be regarded as qualitative observations since they were not based on quantitative physical measurements. The committee received testimony (Martin, 2010) stating that some Dugway preparations, particularly those utilizing lyophilization but no dispersant, gave products with similar appearance and electrostatic dispersibility as the letter samples, suggesting that these properties were not necessarily connected to an intentional effort to increase dispersibility through addition of a dispersant. Exogenous silicon and bentonite, which enhance the dispersibility of spore preparations, were not found in the Leahy and Daschle letters.

Note that this analysis provides the strongest evidence to date that the spores used in the attacks did not come directly from the RMR-1029 flask, because the spores in the flask do not have the silicon content as those in the attack material. Note also that growth in the presence of polydimethylsiloxane-containing antifoam agents is seen as one route that needs to be investigated for how silicon can be incorporated at elevated concentration in the spores, just as I suggested in this post.

It is also worth noting that highly purified spores produced at Dugway did have the aerosolizing quality of the attack material. No additional treatments besides purification were needed for these spores to disperse in air, so I suspect that is one of the primary reasons that the NAS panel could not support the FBI claim that someone with a very high level of expertise must have prepared the material used in the attacks.

Rush Holt Blasts FBI for Withholding Documents from Outside Review of Scientific Work in Anthrax Investigation

5:07 am in Uncategorized by Jim White

The infamous RMR-1029 flask genetically linked to the anthrax attack material.

[Ed. note: Link to Rep. Holt's statement and letter have been added below.]

Both the New York Times and McClatchy report that Congressman Rush Holt (D-NJ) has written a letter to the FBI, blasting them for requesting a delay in the release of the final report from the National Academy of Sciences panel that has been reviewing the scientific analyses used in the FBI’s Amerithrax Investigation of the 2001 Anthrax mailings. It appears that in requesting the National Academies to delay release of its final report, the FBI has released an additional 500 pages of documents to the panel, but only after having seen the draft final report from the panel.

Here is McClatchy discussing Holt’s letter (which I don’t see posted on Holt’s website; I will call and request a copy Link to PDF letter here):

Holt, a scientist and the chairman of the House Select Intelligence Oversight Panel, said the academy recently shared with the bureau its draft report on the “Amerithrax” investigation, a narrow scientific review that the FBI requested in 2008 in an effort to quell controversy over its findings that a disgruntled government scientist was behind the attacks.

“This week I was informed by the NAS that the FBI would be releasing an additional 500 pages of previously undisclosed investigative material from the Amerithrax investigation to the NAS,” he wrote. Holt said he understands that the “document dump . . . is intended to contest and challenge the independent NAS panel’s draft findings.”

“If these new documents were relevant to the NAS’ review, why were they previously undisclosed and withheld?” Holt wrote. He requested a meeting with the FBI director.

In the Times, Scott Shane reports that the National Academy has agreed to extend its study:

E. William Colglazier, the academy’s executive officer, said the F.B.I.’s request was a surprise and came after the bureau saw the panel’s peer-reviewed final report, which was scheduled for release in November. He said that the committee’s 15 members, top scientists who serve as volunteers, were “exhausted,” but that the panel had agreed to extend the study and consider revising the report in return for an additional fee, probably about $50,000, beyond the $879,550 the F.B.I. has already paid for the study.

Dr. Colglazier declined to say if the report was critical of the F.B.I.’s work but said it was “very direct.” The report sticks to science and does not offer an opinion on whether Dr. Ivins carried out the anthrax attacks, he said.

The McClatchy article also quotes Holt as saying of the FBI that it “consistently botched and bungled this case from the beginning.” In addition to the early focus on Steven Hatfill as the primary suspect, followed by a settlement of more than $4 million paid to Hatfill after he was cleared of involvement, several aspects of the FBI case do not appear to withstand scientific scrutiny.

In this diary, I pointed out that the amount of highly purified anthrax material that was used in the mailings would have been very difficult, if not impossible for Bruce Ivins, whom the FBI stated was solely responsible for the attacks, to have produced at his government laboratory without arousing the suspicion of his coworkers. The small shake flasks that Ivins would have used produce very little material, so he would have had to grow anthrax in a very large number (over 35 or so) of the cultures he normally grew.

On the other hand, a single production of spores from a fermenter of at least 70 liters would have produced enough anthrax spore material to account for what was used in the attacks. Further, in this diary, I point out that the abnormal silicon content of the spores used in the attack can be accounted for by the presence of an agent called “antifoam”, that is added to microbial cultures when they are grown in large fermenters, again suggesting that the attack material was produced in a fermenter to which Ivins did not have access.

I had been monitoring the website for the National Academies investigation regularly since the project’s slated termination in late October, looking for their final report. It appears now that we all will have to wait a bit longer before we see that report. My only hope is that Rush Holt is making sure that David Margolis is not allowed anywhere near the report before it is made final.

(h/t to @jaraparilla for alerting me to the Times article and to Retired Military Patriot for finding the McClatchy article)

Most Likely Source of Silicon in Anthrax Attack Spores Argues Against Production by Ivins

9:16 am in Uncategorized by Jim White

In "Silicon Analysis of Anthrax Attack Spores: New Answers Leave More Questions Unanswered", I referred to data recently published in Science, where it was found that the anthrax spores used in the attacks of 2001 contained an unexpectedly high concentration of silicon inside them, as a component of the internal spore coat. I also discussed data from a paper by researchers in Japan who demonstrated that they could produce spores with high silicon content using a closely related bacterium by culturing the bacteria in medium containing high concentrations of silicates.

I am deeply indebted to commenter behindthefall (see this comment as just one in the series) in the comments section of the diary linked above for continuing to ask why someone would put a high concentration of silicates in the growth medium. Those persistent "why?" questions kept coming at me, and I finally extended my thinking from just the narrow question of silicates in the medium to think more broadly about any material containing the element silicon which could somehow wind up in the spores. That took me directly to materials called antifoam agents.

Before getting to the silicon content of popular antifoam agents, a brief digression to explain foaming in microbial cultures is necessary. When microbial fermentation is carried out in large fermenters as opposed to small shake flasks, it is common practice to add agents generally classed as antifoams. Microbial growth rate in liquid medium is often limited by the rate of oxygen transfer into the medium. In shake flasks, the flask is filled below the half-way mark and oxygen is supplied simply by swirling the flask with it attached to a moving platform. Oxygen transfer occurs at the liquid-air interface and keeping the liquid circulating in this way allows oxygen to achieve a sufficient concentration in the liquid to support growth. In larger fermenters, on the other hand, the liquid is much "deeper" and so must be both stirred with a mechanical stirrer and aerated through the use of forced air generally introduced at the bottom of the tank, similar to the air pumps commonly used in aquariums.

To appreciate the foam problem that forced aeration induces, consider two different glasses containing a carbonated soft drink. First, consider a highball glass (for you non-drinkers a higball glass ironically has a low profile, just taller than the height of an adult hand and with a similar diameter) filled less than halfway. Swirling this glass gently by hand isn’t going to cause much trouble for containing the liquid. That is the situation seen in shake flask cultures. Now consider a much taller glass tumbler with a narrow diameter and filled to about the three-quarters mark. Imagine that the soft drink is being stirred by a small propeller and you then insert a straw to the bottom of the glass and blow. That is the foamy mess encountered in large fermenters if steps are not taken to control foam.

Antifoam agents work to reduce the surface tension on bubbles, collapsing them.

Although there are multiple types of antifoam agents employed in microbial fermentation, silicone based antifoams are among the most popular. My favorite antifoam agent of all time is Dow Corning Antifoam M (pdf) because in addition to its use in fermentation, it also is used as an antiflatulent.

Here are the typical properties of this material from the Dow website I linked above:

Antifoam M properties

From a biochemical perspective, it seems quite unlikely that the dimeticone itself (polydimethylsiloxane is a large, polymeric molecule with lots of silicon in it) would be able to be taken up by anthrax cells in culture. However, the presence of four to seven percent of the material as silica is quite intriguing, because very small particles of silica carried in the mixture of silicon polymer could be expected to be available for movement into the cells. (In the calculations that follow, I will assume a silica content of 5%.)

That thought prompted a return to the paper from the Japanese researchers to look again at what they had to say about the chemical structure of the silicon they found in spores. It turns out that although they supplied silicon to the cultures in the form of silicates, the silicon inside the spores was most likely present as silica (the "HF" they refer to is hydrofluoric acid, which is a very strong acid that is different from the other "mineral acids" to which the high silicon spores are resistant):

As far as we know, diatoms, plants, and animals accumulate silicate as silica (13). Silica can be dissolved in HF (16). Accordingly, if the Si layer of spores contains silica, it could be removed from the high-Si spores with HF treatment. Approximately 75% of Si that was accumulated in the spores was released as silicate after treatment with 50 mM HF (data not shown). We compared the acid resistance of HF-treated high Si- and low-Si spores (Fig. 7). After HF treatment, the viability of the high-Si spores was no longer higher than that of the low-Si spores. These results indicated that the Si layer mainly contains silica and supports acid resistance.

It seems very likely to me that anthrax grown in the presence of antifoam agents that contain silica would be able to incorporate this silica directly into the spore coat, skipping the step of converting silicates to silica. It appears that typical working concentrations of antifoam agents could achieve silica concentrations in the range at which silicates were incorporated into medium in the experiments in Japan. The silicate concentration in their experiments was 100 micrograms of silicate per milliliter of culture medium. That corresponds to roughly 0.01% of the medium’s total weight in silicates.

Antifoam agents can be effective at very low concentrations. For example, see here for a recommendation for use at 0.005 to 0.02% for the polymer, so for Antifoam M the silica would be only at 0.001% of the weight of the medium, 10-fold lower than the silicate concentration fed in the reported experiments. However, it is common to exceed those low recommended levels. For example, see this publication (pdf) from 1973, where a silicone antifoam was added to a final concentration of 0.5%. In this case, if the agent were Antifoam M, the silica concentration would be 0.025%, well above the 0.01% silicate fed in the experiments in Japan. Also, my own personal experience running a fermentation pilot plant involved many fermentation runs I can recall that added up to a full one percent or more of the total medium volume as polymeric antifoam before the process ended.

If the silicon in the anthrax attack spores does indeed come from the material having been cultured in the presence of a silicone antifoam agent that also had silica present, then the FBI’s conclusion that Bruce Ivins acted alone in the attacks is called into serious doubt. In this diary, I calculated that Ivins would have to have grown 36 of his two liter shake flask cultures to produce the spores used in the attacks. I further quoted pages 26 and 27 of the FBI’s Amerithrax Investigative Summary (pdf):

In 1997, USAMRIID commissioned another Army research facility, Dugway, to prepare large batches of Bacillus anthracis spores for an upcoming series of studies testing the anthrax vaccine, because USAMRIID lacked the capacity to do so. By the fall of 1997, Dr. Ivins received from Dugway seven shipments containing the concentrated product of 12 ten-liter, fermenter-grown lots of Bacillus anthracis – the “Dugway Spores.” By Dr. Ivins’s own account, these spores were not in perfect shape, so he had to “clean them up.” Indeed, he even discarded the seventh shipment because he deemed it to be inadequate. He noted in his lab notebooks the process that he used to clean them, and also sent e-mails to various people noting his frustration that he had to wash them. To the Dugway Spores, Dr. Ivins added concentrates of 22 two-liter batches of spores which he himself prepared with the help of a laboratory technician. He combined his spores with those from Dugway, and put them in two flasks, labeled “GLP [Good Laboratory Practices] Spores.” In addition, he created a Reference Material Receipt record on which he made the following notation: “Dugway Proving Ground + USAMRIID Bact’D – highly purified, 95% unclumped, single refractile spores.” Finally, in his laboratory notebook 4010, page 074, he described the end-product of these efforts as “RMR-1029: :99% refractile spores;

The alternative explanation to Ivins growing 36 two liter cultures is one fermenter run of approximately 70 liters or more. Note that the FBI investigative summary informs us that Dugway was engaged for the 1997 work precisely because Ivins did not have access to large scale culture equipment. The fact that the RMR-1029 spores themselves did not have a high silicon content could be explained by the use an antifoam agent that did not have silica present for those particular fermenter runs at Dugway, since silica is not uniformly found in all antifoam agents. However, the presence of high silicon in the attack spores strongly suggests that they could have been grown in the presence of an antifoam agent that did contain silica. If Ivins had grown the spores in his shake flask equipment, he would have had no reason to include any sort of antifoam agent, much less one containing silica, because antifoam is just not used in shake flasks. It also seems unlikely that Ivins would have changed his culture process to produce the attack material. If he did not introduce silicon in his early shake flask cultures (and we know he didn’t from the silicon analysis of the RMR-1029 material), it seems unlikely he would have done so with shake flasks for the attack material.

Note also from the Science report that the only other elevated (but not as high as the attack spores) silicon content spores analyzed came from Dugway, where we know that fermenters are available.

In conclusion, the finding of high silicon in the spores used in the anthrax attacks suggests that these spores were grown in a large fermenter that used an antifoam agent containing silica. Since Bruce Ivins did not have access to a large fermenter, fermenter growth would suggest that he could not have acted alone in the attacks.

This hypothesis could be tested easily in a series of experiments where B. anthracis or B. cereus is grown in media with a range of concentrations of antifoam agents with and without silica present in them. followed by analysis of the silica content of the spores. From an investigation standpoint, it would not be difficult to determine if Ivins or someone in his laboratory ever purchased an antifoam agent containing silica that could have inexplicably been used in shake flasks.

Update: Due to the ongoing conversation in the comments below, it is useful to see the analysis of silicon locations in the high silicon spores in the Japanese study cited above. In the illustration below, CX stands for cortex, CT for coat, SX for particles containing silicon, EX for exosporium and UC for undercoat:

Japanese high silicon spore

To my eye, this silicon coating of the spore coat looks just as contiguous as that in the electron micrographs of the attack spores in the previous diary.

Silicon Analysis of Anthrax Attack Spores: New Answers Leave More Questions Unanswered

1:00 pm in Uncategorized by Jim White

RMR-1029 Flask

We now know conclusively that the spores in the anthrax attacks did not come directly from this flask labeled RMR-1029 by Bruce Ivins, but were cultured most likely using RMR-1029 as the culture inoculum.

Among the many enduring scientific mysteries surrounding the Amerithrax investigation of the anthrax attacks of 2001 is the question of whether an agent such as fumed silica was used to "weapoinze" the spores. Early in the analysis of the attack, statements were issued claiming that the spores were indeed weaponized with silica, but subsequent analysis has proven that not to be the case. New data just released help to pinpoint the reason for the earlier misunderstanding while at the same time emaphasizing that central questions about the conclusion that Bruce Ivins acted alone remain unanswered.

To review, here is a key Washington Post article from October, 2002 that fed into the "weaponized" theory:

A significant number of scientists and biological warfare experts are expressing skepticism about the FBI’s view that a single disgruntled American scientist prepared the spores and mailed the deadly anthrax letters that killed five people last year.

These sources say that making a weaponized aerosol of such sophistication and virulence would require scientific knowledge, technical competence, access to expensive equipment and safety know-how that are probably beyond the capabilities of a lone individual.

/snip/

Several sources agreed that the most likely way to build the coated spores would be to use the fine glass particles, known generically as "fumed silica" or "solid smoke," and mix them with the spores in a spray dryer.

Read the rest of this entry →

Could Ivins Have Produced All of the Anthrax Spores Used in the Attacks?

8:50 am in Uncategorized by Jim White

RMR-1029
Could Bruce Ivins have produced his own large batch of pure anthrax spores?

In a previous diary, I examined the information presented by the FBI in their Investigative Summary (pdf) that was published in closing the Amerithrax investigation of the anthrax attacks of 2001. After first mistakenly assuming too small an amount of anthrax powder in the attack letters, I later corrected the diary to reflect agreement with the FBI’s statement from an anthrax expert that the spores in each attack letter would have come from the equivalent of 100 mL of the infamous flask labeled RMR-1029 that was in Ivins’ possession. This diary will work forward from that basis to ask the question of whether Ivins could have produced the attack material at the USAMRIID facility.

Here is the passage from pages 26 and 27 of the Investigative Summary where the production of RMR-1029 is described:

In 1997, USAMRIID commissioned another Army research facility, Dugway, to prepare large batches of Bacillus anthracis spores for an upcoming series of studies testing the anthrax vaccine, because USAMRIID lacked the capacity to do so. By the fall of 1997, Dr. Ivins received from Dugway seven shipments containing the concentrated product of 12 ten-liter, fermenter-grown lots of Bacillus anthracis – the “Dugway Spores.” By Dr. Ivins’s own account, these spores were not in perfect shape, so he had to “clean them up.” Indeed, he even discarded the seventh shipment because he deemed it to be inadequate. He noted in his lab notebooks the process that he used to clean them, and also sent e-mails to various people noting his frustration that he had to wash them. To the Dugway Spores, Dr. Ivins added concentrates of 22 two-liter batches of spores which he himself prepared with the help of a laboratory technician. He combined his spores with those from Dugway, and put them in two flasks, labeled “GLP [Good Laboratory Practices] Spores.” In addition, he created a Reference Material Receipt record on which he made the following notation: “Dugway Proving Ground + USAMRIID Bact’D – highly purified, 95% unclumped, single refractile spores.” Finally, in his laboratory notebook 4010, page 074, he described the end-product of these efforts as “RMR-1029: :99% refractile spores;

One of the footnotes to the paragraph above reads as follows:

Although he received 164 liters in total, once combined, the spores themselves were harvested and resuspended in a total of approximately one liter of liquid, divided into two one-liter flasks. At some point, when the volume was sufficiently depleted, Dr. Ivins combined the spores into one flask. See Attachment E.

As noted in the previous diary, RMR-1029 was at a concentration of approximately 3 X 1010 spores per mL, or for a one liter batch, there were approximately 3 X1013 spores on hand before any spores were used in experiments. The footnote reproduced above states that Ivins received 164 L of culture material to produce the final, cleaned spores, but this number reflects adding together the 12 ten L batches from Dugway with his own 22 two L batches that he produced. It does not allow for the one shipment out of the seven received from Dugway that Ivins discarded. Since the calculations I am trying to carry out here are aimed at how many L of anthrax Ivins would need to grow to produce the materials in the attack letters, I will assume that the discarded shipment represented 2 ten L fermenter batches from Dugway. (Seven shipments representing 12 ten L batches could be explained most easily if five shipments had two batches and the remaining two each had one batch.)

Therefore, if only 10 of the Dugway ten L fermenter batches were combined with Ivins’ 22 two L flask cultures, then a total of 144 L of anthrax cultures were cleaned and concentrated to the final one L of RMR-1029. For five attack letters with an equivalent number of spores to those contained in 100 mL of RMR-1029 each, that would mean that Ivins would have to have carried out 36 two L cultures to produce the equivalent number of spores as half of RMR-1029. Note the beginning of the first quoted passage above: Dugway was asked to produce their fermenter lots of anthrax "because USAMRIID lacked the capacity to do so".

Two L cultures were most likely the largest individual cultures Ivins could produce. After all, if he could have grown cultures of four or eight L each, he would have needed to grow fewer of them when he was producing his own material to add to RMR-1029. There is also a more mundane reason two L would be his upper limit. Without access to a fermenter, the largest culture flasks microbiologists commonly use are 2.8 L flasks commonly referred to as Fernbach flasks, as seen on the right in this photo. Although a larger flask is shown on the left in the photo, the Fernbach style is preferred because of its low profile. Bacterial growth rates for aerobic (oxygen-consuming) bacteria like anthrax are dependent on oxygen transfer which occurs at the interface between the growth medium and the surrounding air. Although it appears that the flask on the left is filled over the two L mark, such an arrangement would be unsuitable for culture because of poor oxygen transfer at the higher, narrower level of the Ehrlenmeyer flask. Fernbach flasks are not filled above the two L mark for similar reasons.

The flasks in the photo linked above are attached to a standard bacteriological shaker. Note that the "holders" on these shakers are interchangeable, but they commonly are not available in sizes larger than those shown in the photo. Also note that the large flasks take up a lot of room on a standard shaker. It would be expected that USAMRIID would have a large number of shakers (and shakers are available with larger platforms than the one in the photo), but it is very difficult to imagine how Ivins could carry out 36 of his two L cultures in a short time, especially within the time frame in which the FBI notes Ivins’ extended hours in the laboratory. Similarly, it would seem that so many large cultures being grown in a short time would run the risk of crowding out shaker space for other researchers in the facility, and the FBI does not mention other researchers noting excessive shaker use.

Footnote 10 on page 27 of the investigative summary notes that at most, 200 mL of RMR-1029 is unaccounted for. If we assume that material was used in the attacks, then the need for culturing is dropped to the equivalent of 300 mL of RMR-1029, which would still be about 22 of the two L cultures.

Here is the relevant passage from page 30 of the Investigative Summary explaining Ivins’ extra hours in the "hot suite" where anthrax could be safely handled:

However, beginning in the middle of August 2001, there was a noticeable spike in his evening and weekend access to B-313, which continued in spurts through October 2001, and then trailed off to his typical pattern. The data for 2001 revealed the following: January through July: eight hours and 48 minutes total in B-313 during off-hours; August: 11 hours, 15 minutes; September 2001: 31 hours, 28 minutes; October: 16 hours, 13 minutes; November: six hours, 20 minutes; December: three hours, four minutes. (See Attachment H; see also Attachment I, depicting Dr. Ivins’s off-hours lab access over four years). There was no big experiment or project going on in September/October 2001 that would justify all of the time in the hot suites. Even Dr. Ivins could not explain this extraordinary change in his work schedule.

Although the Investigative Summary, in discussing the off-schedule hours for Ivins, notes "He had unfettered access to the necessary tools to grow, harvest, and purify the anthrax, as well as to the equipment capable of performing the forbidden function of drying the anthrax" the subsequent discussion of time spent appears to be dedicated to the process of purifying and drying the spores. The production of the spores themselves is only addressed in that passing "grow" in the sentence above. Yet, as seen in this paper (pdf), the growth cycle for the vegetative (non-infectious) stage of anthrax requires at least an overnight culture (Figure 1 of the paper suggests about a 16-24 hour period needed for growth). Furthermore, a shift in growing conditions (and most likely, growth medium) is needed to achieve sporulation. Figure 2 of this paper suggests a sporulation time of another five to eight hours.

In short, the discussion in the FBI’s Investigative Summary appears to me to be able to account for Ivins’ late hours just before the attacks as devoted solely to the final stages of purifying and drying the spores involved in the attacks. For example, in discussing the abnormal time entries for September, footnote 17 on page 31 of the summary states:

Numerous microbiologists have concurred that two hours and 15 minutes would be enough time to dry Ba spores, depending on factors such as the quantity of starting material, the volume of liquid in which it was suspended, and whether a centrifuge was used to eliminate most of the water, leaving behind a pellet, or paste, capable of being dried in well under two hours.

However, accounting for the purifying and drying of the spores ignores the time that would have been devoted to growing them. I don’t see how the large number of two L cultures that would have been needed could have been produced in these same time slots. The flasks would need to be on the shakers over 24 hours for each batch, and that means other workers in the facility would have noted them. Since there is no mention of workers noticing a large number of abnormally large culture flasks on the shakers during the time frame in question, one is left with postulating that Ivins planned the attack much earlier and grew the large number of two L cultures one or two at a time. This process would have required months to grow the spores and combine the crude, first harvest of spores that were then processed in September and October of 2001.

Although the Investigative Summary does not mention it, one theoretical way to overcome absence of evidence for Ivins producing a large number of cultures would be if Ivins made a technological improvement in either the yield of spores from the culture process or the yield from the cleaning process. Also, the calculations here are assuming that the yields from the original cultures that contributed to RMR-1029, the fermenters at Dugway and Ivins’ flasks, were the same. The Investigative Summary provides no evidence on that front, although it is likely that information is present in Ivins’ notebooks, as he appears thorough enough to have recorded volumes received and spore concentrations in each batch shipped. The overall yield for clean spores in RMR-1029 comes out to 2.1 X 10 11 per L of original culture, averaged over the 144 L of culture that contributed to it. That is not a particularly high bacterial yield, so improvement by a factor of ten could be considered as feasible. With such an improvement, and with the use of the missing 200 mL of the original RMR-1029, Ivins might have needed only about 2 or 3 two L cultures to produce the rest of the needed material in the attacks. Those might be grown in the facility unnoticed, but he still would have had to be especially efficient in his time spent in the hot suites to produce them.

From the very large number of spores used in the anthrax attacks, it is much easier to believe that the material was produced in a fermenter. Although Dugway produced 12 different ten L batches for RMR-1029, and would need about 7 ten L batches at the average yield amount to account for the attack spores, fermentation facilities have access to larger fermenters. Only one surreptitious batch of about 70 L or more would be required to produce the attack material. Both Dugway and Batelle (referred to in the report as a commercial facility in the midwest, see page 35) had access to RMR-1029 and have the needed equipment to produce such a large batch of spores. How carefully did the FBI investigate the possibility of fermenter batches at these facilities? A single large fermenter batch seems to me a much more likely event than Ivins being able to hide the production of 36 two L cultures in a short time, develop a 10 fold improvement in the process, or hide the initial culturing of spores over a number of months prior to the attacks.

In short, the FBI has provided a feasible account of how Ivins could have dried the spores and loaded them into letters, but it has not provided an adequate account of how he was able to culture such a large number of spores without being detected.