|Many of Dr. Bruce Ivins' friends and
associates (and lots of conspiracy theorists) have argued that Dr.
Ivins couldn't have made the approximately three trillion spores used
in the anthrax attacks of 2001 without someone noticing it and
questioning him or notifying security that Dr. Ivins was doing
There's ample evidence, however, that Dr. Ivins did things routinely that should have been considered "suspicious," but no one paid any attention. Moreover, the facts say that the spores used in the anthrax attacks of 2001 were very likely created in plain sight, under the noses of Dr. Ivins' friends and co-workers, and would have appeared to them to be part of Dr. Ivins' normal work.
Dr. Ivins' Evening Hours
Dr. Ivins often entered Building 1425 at USAMRIID in the evening, so his presence wouldn't have attracted any attention by the guards. They were accustomed to seeing him around after normal work hours. Mostly, Dr. Ivins would go to his office and use his computer, browsing the Internet and sending emails to former friends and associates - particularly to Mara Linscott, who had been one of Ivins' assistants until she left to go to medical school in July of 1999.
However, Dr. Ivins also occasionally used his lab in Bacteriology Suite 3 during the evening. The differences starting on August 31, 2001, were that he began working alone in his BioSafety Level-3 laboratory every night, and he worked many more hours per evening than he'd worked during past evenings. (See my web page on "Where & When Bruce Ivins Made the Anthrax Powders" for further details.)
The Source of the Spores
The facts appear to indicate that the Ames strain anthrax used in the letter attacks of 2001 began with either the July 9 or the August 27, 2001 withdrawals of spores from flask RMR-1029, which Ivins controlled. Dr. Ivins logged the withdrawals on page 2 of the Reference Material Receipt Record:
Here's an unredacted version of the entries showing that the August 27 withdrawal was for Dr. George Ludwig of the Diagnostic Systems Division. The July 9 withdrawal appears to be for use in Suite B3:
But, the spores that were taken from the flask weren't directly turned into the anthrax powders.
The Guinea Pig Experiment
Ten weeks prior to the anthrax tests, 91 guinea pigs had been vaccinated with 4 different substances: 63 were vaccinated with the standard AVA vaccine and two variations on that vaccine, and 28 guinea pigs were vaccinated only with a saline solution for control and comparison purposes.
Starting around September 1, all 91 guinea pigs were each injected with about 5,000 Ames anthrax spores.
On September 10, 2001, Dr. Ivins wrote this email about the results:
So, after 10 days the test results were:
28 guinea pigs had been vaccinated only with a saline solution. All but 1 died.
32 guinea pigs had been vaccinated with the AVA anthrax vaccine. 15 survived.
15 guinea pigs had been vaccinated with a variation of the AVA vaccine. 12 survived.
16 guinea pigs had been vaccinated with a different variation of the AVA vaccine. 11 survived.
91 guinea pigs had been vaccinated and then injected with anthrax spores for the test
Preparing the Spore Injections
The anthrax spores used in the injections couldn't be used directly from the flask. The spores from flask RMR-1029 first had to be diluted with distilled water, divided into at least 91 separate portions and then each portion had to be tested to make certain that each animal would be injected with approximately 5,000 spores.
Page 18 of FBI pdf file #847406 says about the process of serial dilutions:
In XXXX, USAMRIID began
using rabbits in Anthrax studies instead of mice. The quantities
used were between three hundred (300) and four hundred (400)
rabbits per study. XXXXXXXXXXX stated that rabbits were used more
often than primates were used in Anthrax challenges. Challenges
were run three (3) days per week with thirty (30) rabbits per day.
Two (2) dilutions of Anthrax were prepared per rabbit for each
challenge, and three (3) Tryptic Soy Agar (TSA) plates were
cultured per dilution. XXXXXXXXXXX noted that a total of
approximately 180 TSA plates were cultured per day when animal
challenges were being conducted. The TSA plates were obtained from
the XXXXXXXXXXXXXX group at USAMRIID.
Assuming that similar preparations were done for subcutaneous injections of guinea pigs, that would mean that each guinea pig required two dilutions of anthrax and three Tryptic Soy Agar (TSA) plates per dilution. In other words, 6 plates were needed to test the solution that would be injected into 1 guinea pig, and for 91 guinea pigs, 91x6 or 546 plates would be needed. A fixed amount of diluted spores and liquid would be used to inoculate each agar plate, spreading the spores around on the plate, and the plates would then be put into an incubator overnight. In less than an hour after the start of incubation, the spores would start to germinate and turn into living bacteria. The bacteria would then begin to replicate. Each viable spore would form a "colony" of growing bacteria on the surface of the agar. The next day, the number of anthrax colonies on the plates would provide an approximation of how many spores were actually in the original fixed quantity of liquid.
This was all normal, routine work for Dr. Ivins and was almost certainly done by him and/or his assistants during normal daytime work hours. There was nothing being done that would raise any suspicions. The next day, if any of the plates showed too many colonies or too few colonies, the amount of spores in an injection sample would have to be adjusted by adding more liquid or by starting fresh with a new fixed quantity. And, more plates would be created with more colonies.
The end result after a few days of preparations would be correct amounts of spores in each syringe and many hundreds of inoculated agar plates which were tossed into autoclave bags for sterilization and disposal.
That's where things stopped being "normal." In most labs, it would be "normal" to put the autoclave bags into an autoclave at the end of the day and sterilize the contents. Then the sterilized contents would be removed from the autoclave and disposed of (or set in a place for disposal by the janitorial staff).
In Dr. Ivins lab, what was "normal" was a bit different from what was "normal" elsewhere.
FBI pdf file #847373, page 3, says:
no consistent method for
the disposal of
was documented during several
interviews that Ivins'
said that he had seen the
XXXXXXXXXXX stated that some of
the post-challenge plates
After the plates had been
left in the incubator
Traces of Agar
The facts say that the spores used in the anthrax attacks of 2001 came from the colonies that continued to grow on the hundreds of plates that were inside those autoclave bags. The colonies grew until they covered the plates and consumed all the agar. Then, without any nutrients to continue growth, the bacteria formed spores. The facts confirm that the attack spores were not produced in shaking flasks or a fermenter.
FBI/NAS file B1M1 page 25:
Based upon evidence recovered from the letters it was the group's consensus that the material was likely produced in small batch or batches by someone skilled in the art of microbiology. Due to the propensity of contamination in liquid preparations, even by those very skilled in the art, it may suggest an agar-based production.
The single color Daschle material is suggestive of careful removal of the top layer observed in the centrifugation of anthrax preparations (by USAMRIID) while the tri-colored material in the NY Post letter is suggestive of the use of all three observed layers or an air-dried preparation.
FBI/NAS file B3D1 page 35:
During the week of 7/8/02 UMD [University of Maryland] also detected the presence of agar in the New York Post evidence and a trace of agar in the Leahy evidence.
FBI/NAS file B3D1 page 169:
Carbon-13 data from more than 300 media samples suggests that the evidence was grown on a C3 type media. There are two biochemical pathways, C3 and C4, by which plants convert CO2 to carbohydrates. C3 nutrients are derived from either C3 plants, such as cool weather grasses, or from animal tissue that has grazed on such plants. Many common C4 media are derived from North American beef extracts and cattle products to include casein (mild protein) digests. C-4 plants and related animal products include the "nutrient" type media derived from corn fed animals, typically pigs, and digests of gelatin, which are usually hydrolysis of of pig parts. Yeast Extracts provide yet a third class of isotopically distinguishable media profiles. Data from earlier carbon dating analysis, indicates that a C3 growth media was used to culture the evidence.
FBI/NAS file B3D1 page 191:
The isotopic analysis of the Leahy evidence is complete. The C-13 results are consistent with the use of C-3 plant/animal type media. The O-18 data suggest an isotopic enrichment beyond that expected from typical microbiological growth conditions or from any known water source. Such enrichment could occur from evaporation (growth plates left open to the air) or an extended growth time beyond the normal 2-3 day growth used by microbiologists.
FBI/NAS file B3D1 page 245:
Furthermore, the NY Post material, known to contain 6% agar and other debris, may still have traces of heme present, if blood media was used in its production.
FBI/NAS file B1M9, pages 32 through 44 are a February 22, 2004 report titled "Stable Isotope Characteristics of Anthrax Sample SPS 02.266." (SPS 02.266 is the Leahy spores.) The report is by Drs. James Ehleringer and Helen Kreuzer-Martin of the University of Utah. Page 34 says:
With these important limitations, the isotope ratio data from sample 02.266 are
* inconsistent with the spores having been produced with water from Dugway Proving Ground
* inconsistent with the spores having been grown in liquid medium made with any known meteoric water source
* consistent with the spores having been grown on solid medium, perhaps for an extended period of time.
So, the findings by Ehleringer and Kreuzer-Martin show the Leahy spores probably weren't from Dugway, weren't grown in a liquid medium, and appear to have been grown on "solid medium" agar plates over an "extended period of time."
On page 77 of the NAS report reviewing the science used in the Amerithrax Investigation, they used a figure of 8 billion spores per plate (8 x 10 to the 9th), and they calculated that meant that the anthrax mailer would have required a minimum of 463 plates to make all of the anthrax spores in the letters.
If the 91 guinea pigs used for the subcutaneous Ames strain tests that began on September 1, 2001, required the same number of plates, that would compute to two (2) dilutions of Anthrax per guinea pig and 3 TSA plates per dilution. Or:
91 x 2 x 3 = 546 plates ... more than enough for the attacks
That means that the attack spores almost certainly came from agar plates inoculated with spores from flask RMR-1029 that were allowed to grow until all the media was consumed and the plates were entirely covered with growth. Plus, the spores were produced at lower temperatures, slowing the process of sporulation and causing the absorption of silicon into the spore coats from the growth media. And, the only place where that was routinely done at USAMRIID was in autoclave bags that were allowed to lay around for weeks in Bruce Ivins' lab before they were put into the autoclave, plus other bags that Ivins allowed to remain inside the autoclave for long periods of time before the autoclave was turned on to sterilize the materials inside.
Making Crude, Dry Powders
Inside the autoclave bags setting in his lab, Dr. Ivins had all the spores needed for the anthrax letters. All that was needed was to turn into powders all the spores that were loose in the plates and the spores that were captured in matrix material (i.e., slime resulting from enzymes breaking down the carcasses of the dead mother bacteria that produced the spores). And Dr. Ivins had all the necessary equipment for that task in his BSL-3 lab in room B313. Here's a photo of him handling stacks of plates in his lab:
The first step in creating the dry powders was to remove the spores from the plates. This may have been done by simply scraping the spores, slime and debris out of the plates and into a beaker, but a small amount of distilled water may have been used help get the spores out of the plates and into the beaker. The work would have been done inside a biosafety cabinet, like the one seen behind him in the photo above, to prevent contamination of the lab in the event that any of the spores become aerosolized.
During the process of scraping spores into the beaker, Dr. Ivins may or may not have noticed that one of the test plates was contaminated with Bacillus subtilis spores. If he noticed, he may have figured that it would cause investigators to conclude that the spores he had made were created under crude, unsophisticated conditions, perhaps even in someone's basement or garage instead of a laboratory. The contamination would further indicate a crude process performed by non-expert, Muslim terrorists.
Once he had enough crude material for the five media letters in the beaker, Ivins would have begun the process of centrifuging the material to get rid of excess water and other liquids. Below is a photo of centrifuge tubes inside a centrifuge:
Centrifuging would separate most of the spores from the slime, carcass pieces and leftover agar. The results would be concentrated into separate layers in the centrifuge tube:
For the media letters, it appears that Dr. Ivins removed the top three layers from the tube, spread the still-wet material on a plate and allowed it to air dry inside a biosafety cabinet - perhaps adding some heat to speed up the drying process. Depending upon how much water was still left in the material, the material could have easily dried in 2 to 2½ hours. One plate per letter would have been sufficient.
While the centrifuging and drying processes were going on, Dr. Ivins evidently continued scraping spores out of the rest of the hundreds of plates and putting the material into a second beaker.
Tests by investigators showed that the New York post powder contained 6% dried agar, another 84 % was evidently a combination of dried slime, dead mother germs and partial carcasses. Only about 10 percent of the media powders were actual viable Ames anthrax spores. Here's what the New York Post powder looked like under high magnification in a Scanning Electron Microscope (SEM):
Under even higher magnification, some of the New York Post powder clearly showed countless spores embedded in dried slime:
As stated above, the Post powder reportedly showed three distinct colors, which was almost certainly the result of taking all three top layers out of a centrifuge tube. The top layer would have been nearly white and would likely have been almost pure spores. The second layer, which would have been light brown in color, would have been dirty spores, i.e., spores covered with matrix slime and debris (agar particles and parts of the carcasses of dead mother germs). The darker brown third layer would have been mostly matrix slime and debris.
Below is a photograph of how the New York Post powder looked to the naked eye:
Although the powder may have looked very similar to beach sand, there were still plenty of individual spores and clumps of two or three spores small enough to seep through the envelopes and infect people. But, it would take awhile for the victims to begin showing symptoms, and, in most cases, it would take even longer for the symptoms to be correctly diagnosed.Between Mailings
Dr. Ivins had undoubtedly expected the five anthrax letters he dropped into a Princeton, New Jersey, mailbox on the night of September 17 to create headlines across America, and possibly (or hopefully) even panic. But nothing happened. After ten days, when it was clear that the media letters had not achieved their purpose, Dr. Ivins' in-out logs indicate he began work on a more sophisticated batch of anthrax powder using the contents of the second beaker: the remainder of the spores he had scraped from serial-dilution plates.
Dr. Ivins clearly wanted to make certain that no one would be able to ignore the second mailing. The letters he was going to send to Senators Daschle and Leahy wouldn't contain the crude powder which seemed to have been ignored by everyone. The second powder would consist of purified spores -- two and a half trillion spores per gram, close to the theoretical limit. And the powder would also not be contaminated with Bacillus subtilis spores. It would be the scariest batch of anthrax spores Bruce Ivins knew how to create. The process of purifying the anthrax spores was a task Ivins had performed countless times during his decades of work at USAMRIID. The process began with the same step used for the first batch: centrifugation.
This time, instead of removing all three layers from the centrifuge tube, Ivins would carefully take out only the top layer -- the layer of pure anthrax spores -- and he'd set that layer aside.
Then he'd remove the second layer of mixed spores, slime and debris from each of the centrifuge tubes. He would put that material into a small flask of water. The slime and debris from the third layer and the water below it would be autoclaved and disposed of.
The spores and slime from the second layers would be mixed with clean, distilled water. The mixture would be thoroughly shaken (by hand) in an attempt to wash off as any particles of agar or slime clinging to the spores as possible. After this "washing" step, the material would be poured into centrifuge tubes and centrifuged again. The results would - in concept - would look something like this:
Again, the top layers of pure spores would be removed from each centrifuge tube and set aside. The second layers would be put in clean distilled water, shaken, divided into clean tubes and centrifuged again. Everything else would be autoclaved and disposed of.
This process would be repeated as many times as necessary to produce the maximum number of pure, clean spores by washing away as much of the slime and agar particles as possible.
When Ivins was satisfied that he'd gotten what he needed, the next and final step was to dry the pure spores he'd set aside. Dr. Ivins didn't have access to a spray dryer, and using the lyophilizer (freeze dryer) in Suite B5 was out of the question, since there was no way to get the refrigerator-size machine into Suite B3 without attracting attention and without thoroughly contaminating the lyophilizer. Chemical drying was another technique, but it might leave behind chemical evidence that could be traced back to his lab. That left just the simplest method: air drying -- the same method he used for the first batch.
The Leahy Powder
When the powders were dry inside the biosafety cabinet, Ivins would have scraped the dried powders off the plates and into the two letters. The powders would probably have looked like tiny, crumbly flakes of off-white paint as Dr. Ivins folded the paper around the powders - using the pharmaceutical fold to help prevent any powder from escaping the folded sheet of paper. After putting the letters into the envelopes, he taped the flaps shut, adding extra tape in the open corners where letter openers are inserted. He didn't want any of the powder to escape the envelopes before they reached their destinations - the offices of Democratic Senators Daschle and Leahy.
Dr. Ivins evidently didn't consider the handling that would take place as the letters made their way through the machines used by the postal system. The letters went through machine after machine, each machine crushing the flakes inside into smaller and smaller pieces. The machines also squeezed the envelopes and cause any air inside to push through the tiny pores in the paper. Individual spores escaped through the paper of the letter and the paper of the envelope to contaminated postal facilities along the way. The escaping powders also cross-contaminated the outer surfaces of other envelopes which went through the postal machines around the same time. Billions of individual spores escaped that way. But, most of the powders remained in clumps. This is a SEM photograph of a clump of spores from one of the senate letters:
Below is a closer view of such a clump. This clump is known to be from the Leahy letter:
And the picture below shows what the Leahy powder looked like to the naked eye after it had been removed from the letter:
The way Dr. Ivins used the pharmaceutical fold to wrap the powders inside the letters, the way he taped the open corners of the envelopes shut, and the way he included medical advice in the first batch of letters ("TAKE PENACILIN NOW") and told recipients of the second batch of letters that the powder was anthrax ("WE HAVE THIS ANTHRAX") says that Ivins didn't intend for anyone to die as a result of what he'd done. But, five innocent people did die, and 17 others were infected but survived.
The facts indicate that scientists working with the FBI were able to determine that the attack spores were produced from bacterial growths on agar plates and almost certainly from plates on which most of the anthrax bacteria had been allowed to grow at room temperatures, as would happen inside autoclave bags awaiting sterilization. An FBI agent discussed this subject with Dr. Ivins in a telephone conversation on April 15, 2003. Two days later, in another telephone conversation with an FBI agent, Dr. Ivins evidently felt compelled to comment on the method ... and to first point to someone deliberately using the Ames strain because it was "vaccine resistant" and then pointing to Building 1412 as a possible source for the attack anthrax.
This from page 45 of FBI File #847443, dated April 17, 2003:
IVINS feels badly because his work could have played a
role in the anthrax mailings. Someone could have read his research
and determined that Bacillus anthracis (B.a.) Ames was vaccine
After his telephonic conversation with SA XXXXXXX on
04/15/2003, IVINS feels sick over the fact that the material used
in the anthrax mailings could have come from a stock made from the
B.a. aerosol challenge trash.
The facts seem very clear: Dr. Ivins could not explain the long hours he worked alone in his lab shortly before the two attacks. He was known to leave lab materials in autoclave bags in his lab for long periods of time before putting them into the autoclave for sterilization. Thus, he had access to sufficient plates and sufficient spores to produce all the materials in the anthrax letters without attracting anyone's attention by making additional spores from scratch. He had no alibi for the times of the mailings, and the facts clearly indicate that he had no further need to go into his lab for a week or more after he had finished making the powders and mailing them.
Combined with the hidden message in the media letters that directly connected him to the media mailing, various other connections he had to the scene of the crime (the mailbox), and his psychological inclination for revenge against what others had done to him, there really can't be any doubt that Dr. Bruce Edwards Ivins was the anthrax mailer. But, there are still some scientific issues that conspiracy theorists endlessly debate.
Silicon in Spore Coats
The facts seem to indicate that growing spores slowly at "natural" temperatures - as one would find in a pasture where cattle are grazing - will cause the bacteria to utilize silicon from the environment when forming spores. It appears that the silicon can provide some natural protection against UV light. And, the process of absorbing and utilizing silicon appears to require the very slow growth found at less-than-incubator temperatures.
Since we know the plates Ivins used were initially allowed to form colonies in incubators in order to verify the number of viable spores in a sample to be used for an inoculation, in theory that might explain why about a quarter of the spores contained no silicon in their spore coats: Those were the spores grown at incubator temperatures. And since about three quarters of the spores in the letters contained silicon in their spore coats, those could be the spores grown at room temperatures after the plates were tossed into the autoclave bags. That may be an over-simplification, since it is also required that the agar contain silicic acid, plus there are indications that Leighton-Doi media may have played a role in causing the absorption of silicon.
Silicon in The New York Post Powder
An early test of one tiny sample of the New York Post powder found about ten percent of that specific sample was silicon. That unusual reading for silicon was never adequately explained, and unexplained findings are the fodder upon which conspiracy theorists feed. Since the New York Post powder was very crude, logically it could not be silicon from any weaponization process, but conspiracy theorists continue to insist it was.
The facts suggest that the unusual reading more likely came as a result of the fact that the powder was centrifuged before drying. That means the powder was not homogenous. There were chucks of pure dried slime and chunks that were almost pure spores. And centrifugation could have resulted in a concentration of silicic acid from the growth media in one layer within the centrifuge tube. After being dried by Ivins, then crushed in postal machinery, that layer could have ended up as broken in pieces in the powder. A chance examination of such a piece could have found that the piece was 10% silicon. There are probably other possible explanations that are much more logical than that the crude powder had something to do with weaponization utilizing silicon or silica.
The problem with these unresolved issues is, of course, even if you performed experiments and confirmed that silicon would be absorbed into spore coats if allowed to sporulate at room temperatures, or experiments which showed that a layer of silicon would appear in centrifuged material from agar plates, the conspiracy theorists will simply argue that there is no firm proof that that is how the attack anthrax was made. Thus, they feel free to continue their arguments that the silicon shows that the attack spores were made with supersophisticated equipment and a secret weaponization process that the U.S. government is hiding from the public.
Unfortunately, it appears there will always be unanswered questions for which the conspiracy theorists believe they and only they have the true and correct answers.
Meanwhile, during the Cold War, when Dugway Proving Grounds "weaponized" spores by freeze-drying them, milling them, and coating them with particles of silica, here is what a single "weaponized" spore looked like: