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.