Archive for the 'Anthrax attacks' Category

Hypothesis: Brownian Motion can strip artifical anthrax coating

March 19, 2010

Hypothesis: Brownian Motion can strip artifical anthrax coating

The following is a hypothesis to try to explain how anthrax spores with an artificial coating at the time of the anthrax mailings in 2001 may have lost their outer coating.

Coated individual spores have low drag and low mass compared to clumped uncoated spores that have high drag and high mass.  The lower drag of artificially coated spores results in a higher diffusion coefficient for Brownian motion for these artificially coated spores.

Coated individual spores will thus have a higher mean for the velocity distribution from Brownian motion and a higher standard deviation of velocity.  Thus individual coated spores will experience a tail of much higher kinetic energy and momentum than uncoated spores especially if the latter are clumped.

The higher velocity distribution from Brownian motion and whatever else is impinging the spores will over time subject the outer coating of treated spores to experience collisions of much higher kinetic energy as well as spread out over a wider range of energy.

The binding of the outer coating to the spore itself and to silicon and whatever else is inside the spore will thus be subject to a greater stress including those that may find any particular resonances or weaknesses.  This need only find and enlarge holes and cracks
in the outer artificial coating, which was likely never a perfect geometric form in the first place.

Nature did not evolve to preserve such artificial coatings for a long period.  The natural spores themselves experience a low velocity distribution from Brownian motion and
they have evolved to endure this.  But the artificial coating has not evolved from anything and does not inherently need to be stable under the effect of Brownian motion.

Its quite reasonable to believe that the effect of the higher velocity distribution of the artifically coated spores will eventually widen holes and cracks through collisions of coated spores at high velocity in head on collisions until the artificial coating is gone and the spores acquire a low velocity distribution as natural spores have.  Thus over time the artifically coated spores lose the artificial coating.  Silicon is left inside the spores because once the outer coating is stripped, the spores have high drag and can clump so that their velocity distribution falls to that of normal spores. In fact, the additional internal mass simply helps to lower the velocity distribution thus adding to the stability of the spore.

Artificially coated spores with a high mean and standard deviation of velocity together with natural spores with low mean and low standard deviation of velocity from Brownian motion are not a stable equilibrium.  Over time the artificially coated spores transition
to uncoated natural spores.

Irradiation of spores will combine with the effect of Brownian motion as well as the treatment by TEM to subject artificially coated individual spores to a wide range of velocities and interaction energies. Subjecting artificially coated spores that have experienced months or years of the effects of Brownian motion to irradiation and TEM will tend to cover an even wider range of energy impacts and finish off the possibly tenuous remaining binding of the artificial coating to the spore and whatever is inside the spore.  Collisions of coated spores from Brownian motion then have an enhanced ability
to finish the job of stripping the artificial outer coating.  What is left is natural spores on the outside with whatever they contain of silicon and other elements on the inside.

References

http://www.fredericknewspost.com/sections/news/display.htm?StoryID=95744

http://www.google.com/#hl=en&source=hp&q=irradiated+anthrax+Michael+Sandia&aq=f&aqi=&aql=&oq=&gs_rfai=&fp=1dc62da33e2ff469

Irradiation of the spores before analysis by Sandia:

http://www.sandia.gov/mission/homeland/factsheets/Anthrax_Factsheet_Sep9_08.pdf

http://en.wikipedia.org/wiki/M%C3%B6ssbauer_effect

http://www.epa.gov/radiation/sources/mail_irrad.html


How does irradiation kill anthrax?
Irradiation kills anthrax by shattering its DNA and other cellular components. The process for irradiating mail is the same process used to sterilize medical equipment.

During irradiation, an intense stream of electrons (or x-rays if x-ray technology is used) strikes the mail and any anthrax spores it may contain. The radiation dose is very high, about 56 kilograys of radiation, which is approximately 2 million times more than a chest x-ray.

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


The photons which make up visible light have energies of 270–520 yJ, equivalent to 160–310 kJ/mol, the strength of weaker chemical bonds.

http://jjap.ipap.jp/link?JJAP/33/L697/

http://anthraxvaccine.blogspot.com/2009/01/critique-of-chemical-signature.html

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

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

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

http://en.wikipedia.org/wiki/Brownian_motion#History

http://en.wikipedia.org/wiki/Drag_%28physics%29

Brownian motion

In the micron range of particles there is Brownian motion.  Spores are 1 to 3 microns in length and one micron in width.  The original observations of Brown were themselves from living things c. 1820 and his equipment was obviously much weaker and so he likely couldn’t see individual spores of bacillus but instead larger objects, or in any case not smaller.
D = k_B T/b

b linear drag coefficient


In fluid dynamics, drag (sometimes called air resistance or fluid resistance) refers to forces that oppose the relative motion of an object through a fluid (a liquid or gas).

sqrt(2Dt) = sigma

(Its really the velocity distribution we are interested in, not position.)
The silicon coating decreases the drag b.  Thus it increases D and thus increases sigma. So what happens is that the spores coated with silicon have a high velocity from the brownian motion collisions.  This means they are having collisions at high velocity until the coating is knocked off.  what is left is under the spore coat.

http://seminal.firedoglake.com/diary/36075

http://www.anthraxinvestigation.com/Update-History2005.html

http://discuss.washingtonpost.com/wp-srv/zforum/03/sp_iraq_alibek033103.htm


Ken Alibek: Of course they are WMD. The shelf life of these nerve gasses and chemical weapons. These are chemical weapons and they are WMD. The shelf life would be years.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V8W-40S0D9D-2&_user=10&_coverDate=08%2F08%2F2000&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1258163189&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=5d4a6f0158bcce0a37e92fbd299a2b5d

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

http://www.fredericknewspost.com/sections/news/display.htm?StoryID=95744

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

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

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

Search silicon oxide “binding energy” eV

weaponized anthrax “shelf life”

bacillus spore coat exosporium

irradiated anthrax Michael Sandia

silicone polymer structure

silicon coating anthrax “Brownian Motion”

irradiation anthrax

http://images.google.com/images?hl=en&source=hp&q=spore%20coat%20composition&aql=&gs_rfai=&um=1&ie=UTF-8&sa=N&tab=wi

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

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

http://jjap.ipap.jp/link?JJAP/33/L697/

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

http://caseclosedbylewweinstein.wordpress.com/2009/07/22/hunting-the-anthrax-killer-national-geographic-channel-sunday-july-26-900pm/

Recent silicon coating discussion in comments at Meryl Nass

https://www.blogger.com/comment.g?blogID=6592607595936297457&postID=5493194118103137822&isPopup=true

and at Case Closed

http://caseclosedbylewweinstein.wordpress.com/2010/03/18/capture-of-mustafa-hawsawi-and-laptop-with-anthrax-spray-drying-documents/

The outer artificial coating need only be good enough to last a limited period in this instance.  It can have holes and cracks in itself.  It can be loosely bound to the spore or to silicon inside the spore.  Its only enough for the Brownian Motion to produce a velocity distribution that can find and widen the cracks and holes and loose binding to the spore and inside silicon.

The artificial coating gives the treated spore a low drag and thus high mean and standard deviation of velocity from Brownian motion and the randomization of its velocity.  Head on collisions of artificially coated molecules at the upper end of the velocity distribution need only break, crack or widen the holes and cracks and loose binding to the spore and inner silicon to break off the outer artificial coating.

The remaining natural coated spore then retains inside it the artificial silicon but now has a high drag and thus low velocity mean and low velocity standard deviation.  It is thus within the range of velocity distributions from Brownian motion that spores have evolved to survive.

It is unstable to have two different velocity distributions of spores, one artificially coated and one natural.  Over time the high velocity distribution of artificially coated spores will strip the artificial coating by finding its weak points and concentrating the energy of two spores on the cracks and weak links and holes until the artificial coating is striped off and the natural spore with excess silicon inside is left.

This Brownian motion hypothesis thus reconciles initial observations of highly energetic spores in the lab and later observations of no artificial outer coating but silicon left inside.  Brownian motion in the velocity distribution of low drag high velocity artificially coated spores plus time leads to the striping of the artificial coating but leaving silicon inside the spore.   This links the initial lab observations under microscopes of energetic spores and the Senate office buildings being closed and the later observations of no artificial coating of the spores and high silicon inside them.

==Short summary comment left at Meryl Nass blog

The link below considers a lengthy hypothesis to explain how Brownian motion could have striped off an artificial coating of the anthrax spores leaving silicon inside them.

In a nutshell, an artificial coating would produce a low drag coefficient on the coated spore.  This would lead to a high diffusion coefficient and a high mean and standard deviation of velocity of the artificially coated spores by a random walk diffusion process in the velocity of the spores.

Some artificially coated spores would have higher velocities from the combined effect of high mean and standard deviation in velocity.  When they collided head on, they would widen cracks and holes.  The original outer coating may have had its own flaws plus only weakly been bound to the spore or to silicon inside the spore coat.  Over time, the collisions would weaken the bonds.

When the artificial coating is stripped, the drag coefficient goes up and the spore acts like a normal spore with low velocity mean and standard deviation from Brownian motion.  These spores are then stable. But silicon and other elements inside them are trapped and stay in them.

This hypothesis and mechanism reconciles the initial lab observations of high velocity spores with the later observation of silicon inside the spores but no artificial coating outside them.

https://oldatlanticlighthouse.wordpress.com/2010/03/19/hypothesis-brownian-motion-can-strip-artifical-anthrax-coating/

Also at

http://caseclosedbylewweinstein.wordpress.com/2010/03/18/capture-of-mustafa-hawsawi-and-laptop-with-anthrax-spray-drying-documents/#comment-4656

==

Two distributions of spore velocity in the same sample are not stable as a general principle of both quantum mechanics and random processes.  The artificially coated spores can transition to low velocity spores by losing the artificial coating.

This is a one way transition.  Thus over time, we expect to see the artificial coating that causes a high velocity distribution to be lost leaving normal spores but still retaining their extra silicon inside.

The high velocity distribution is created by the artificial coating having a low drag coefficient and thus a high diffusion coefficient and thus a high mean of velocity and a high standard deviation of velocity.   Over time this high velocity, high energy distribution will decay to the normal low velocity mean and standard deviation by the loss of the artificial coat.  The extra internal silicon is then left over as observed.

==

In October 2001, the initial lab observations were of high velocity spores, unlike what the observers had seen before of other spores. This is direct evidence of the high velocity distribution from Brownian motion in the spores in October 2001.   The high velocity of the spores they saw in the test tube and plates in October 2001 could only come from Brownian motion.  It shows the drag coefficient was very low, so the velocity distribution of the spores was high.

==

The exosporium of the spore, the very outside, is not firm and rigid. There is no firm structure to tie onto to resist the high velocity collisions.  Potholes and cracks will develop and widen until the artificial covering is stripped off.

The spore will expand when it becomes a vegetative cell.  We know that this happened, since people died from the anthrax.  This shows the spore was able to break any artificial covering that was around it.  This shows, if there was an artificial covering, it was not a rigid structure encasing the spores in unbreakable bonds.  Instead it shows the typical forces of spore expansion were sufficient to break the artificial covering.

Any artificial covering designed to weaponize has to be a weak one so that the spore can expand and break it and become a vegetative cell.  Thus an artificial covering to reduce the drag coefficient on the outside will be loosely bound to the exosporium and will not be a rigid covering that can’t be broken by the forces the spore can exert by growing.

==

They saw high velocity spore motion in the test tube and on plates in October 2001.  This is direct observation of the high velocity distribution of Brownian motion.   In a sense, the high velocity distribution of the spores observed directly in October 2001 by examination under the microscope is sufficient itself to demonstrate or show a physically different state of the Senate spores from normal spores.  This difference is in having a low drag coefficient and high velocity distribution.  The low drag coefficient implies dispersal.  The high velocity distribution observed directly was the direct observation of dispersal.

This high velocity distribution of spores observed directly under the microscope is in effect a direct observation of a low drag coefficient of the spores.  Thus it shows their dispersal tendency was higher.  This is most easily explained by an artificial covering.  But such an artificial covering has nothing on the exosporium to grab onto with unbreakable bonds.  Instead it will have a loose connection to the spore and possibly to silicon below the exosporium in the spore coat.

==Dxer Comment at Case Closed

DXer said

March 20, 2010 at 9:27 am

No.

I recommend you read the excellent MICROBIAL FORENSICS, Bruce Budowie editor, available for free through your public library’s interlibrary loan.

==Reply to Dxer at Case Closed

The high velocity of the Senate spores were observed directly under the microscope in October and November 2001.  The spores flew off the plate they said.

They have seen normal spores under the microscope.  So the observation of high velocity spores was a scientifically valid observation.

The spores under the microscope were showing a velocity distribution in all directions not one direction.  That is what Brownian motion is, a random walk in all directions.

“Einstein predicted that Brownian motion of a particle in a fluid at a thermodynamic temperature T  is characterized by a diffusion coefficient  D = kBT / b, where kB is Boltzmann’s constant  and b is the linear drag coefficient on the particle ”

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

In Einstein’s 1905 article he looked at displacement directly.  So he did not have a random walk on the velocity.  That came later in the work of Ornstein and Uhlenbeck.

So in October 2001, the scientists saw a high velocity distribution of the spores.  This was not in one direction so it was not some em field in the lab.  Instead it showed that the drag coefficient of the spores was low.  The math is more complicated than the formula above, but essentially, the velocity distribution has a higher mean and standard deviation from the lower drag coefficient.  This is what they observed directly in October 2001.

The higher velocity distribution shows a lower drag coefficient.  That is either the embedded silicon alone or more likely a coating on the outside.  Such an artificial coating would thus mean that coated spores would have high velocity and uncoated ones low velocity.  This is not an equilibrium.  So over time the coating gets knocked off, leaving the silicon in the spore coat trapped.

The outer coating would not have much to hold onto in the spore.  The exposporium of the spore is not a rigid structure suitable for resisting repeated bombardments from high velocity collisions.  Binding to the silicon inside would be tenuous since it was removed spatially from the outside.  Moreover, any artificial covering had to be designed to break when the spore grew back into a vegetative cell which involves expansion.  We know that happened, since people died from the anthrax.

Thus the Brownian motion in velocity mechanism reconciles the direct observation of the high velocity distribution of the spores in October 2001 and the latter observation of no spore coat but silicon still trapped inside the spore coat below the exosporium.

==

http://www.google.com/url?sa=t&source=web&ct=res&cd=12&ved=0CAoQFjABOAo&url=http%3A%2F%2Fwww.physik.uni-rostock.de%2Fmahnke%2Flehre%2Fss07%2Fandris_ornst-ulenb-pr.odp&rct=j&q=Ornstein+Uhlenbeck+1930&ei=WbakS8LpCYX7lwe8gP1y&usg=AFQjCNFR6eQsPw9PZeW63G_JqhVGJd20Lw

This is a mathematical discussion of the Ornstein Uhlenbeck approach.  It shows that the variance of the velocity distribution is inversely related to the frictional drag coefficient and proportional to the variance of the instantaneous Brownian motion and a damping factor from the mean reversion implied by the frictional drag effect.  See around slide 10 and 11 for the final formula.  See slides 3 and 4 for the set up in terms of a drag coefficient.  Low drag coefficient means a low tendency of the velocity to go back to its target, in this setup zero.  A high drag coefficient means a rapid tendency for the velocity to fall.

Coating the outside of the spore to aid dispersion means in effect coating it to lower the drag coefficient.  Its the mathematical definition relative to the formulas of a random walk in the velocity of the Ornstein Uhlenbeck type.

The high velocity of the spores from Senate anthrax was observed directly in the lab under the microscope in 2001.  So this is a direct observation of a low drag coefficient through the OU type formulas as exhibited in the above link.

==

In the formula discussion the velocity is a signed quantity.  So positive velocity along the x axis means moving to higher x values.  Here the mean of the velocity is zero since there is no preference for right over left, or up v down.

If we define speed as the absolute value of velocity, then the speed will not have a mean of zero.  As the variance of the velocity distribution increases, the mean of the speed increases as well.  Thus a low drag coefficient means the mean and variance of the speed both go up.  This means the mean and variance of the speed of the spore are higher the lower the drag coefficient.

So an artificial coating increases the mean and variance of the speed of the spores.  This shows up under the slide as the effect that the spores seem to be moving fast even if its in different directions.  The higher variance of velocity from the lower drag coefficient thus ends up increasing the collision kinetic energy between coated spores, which are sometimes head on, i.e. they have opposite direction of velocity.

Over time these collisions find the weaknesses, joins, holes, cracks, and loose bonds of the outer coating to the spore.  Thus the outer coating becomes full of pot holes and cracks until it breaks off.

In equilibrium, the outer coating will be gone from the spores and all the spores will have high drag coefficient and be in the same distribution of low speed, i.e. low variance of velocity around a mean velocity of zero. This is exactly what the formulas linked to show.

==

Search Ornstein Uhlenbeck velocity distribution.  Or Brownian motion velocity distribution.

Some derivations from a more elementary starting point are given here:

http://ocw.mit.edu/NR/rdonlyres/6E812E2D-BDB4-4AC9-9E91-E57D7DB64056/0/lec14.pdf

Note the orientation is to diffusion on the displacement.  The original OU paper did a velocity distribution and that is gone through at the link cited above.

==Further comment at Meryl Nass in rebuttal to the No of Dxer.

The formulas for the velocity distribution of a spore under from random collisions in a media of smaller particles is given here:

http://www.google.com/url?sa=t&source=web&ct=res&cd=12&ved=0CAoQFjABOAo&url=http%3A%2F%2Fwww.physik.uni-rostock.de%2Fmahnke%2Flehre%2Fss07%2Fandris_ornst-ulenb-pr.odp&rct=j&q=Ornstein+Uhlenbeck+1930&ei=WbakS8LpCYX7lwe8gP1y&usg=AFQjCNFR6eQsPw9PZeW63G_JqhVGJd20Lw

I apologize for the unsightly link, but its the best I could find.

The formulas show that the variance of the velocity is inverse proportional to the drag coefficient.  The drag coefficient just means the tendency of the velocity to revert back to zero from friction.

F = – gamma m v

where m is the mass of the spore, gamma is the drag coefficient, and v is the velocity.  There is an additional random forcing term from random collisions with molecules.

When gamma is low, the drag is low, i.e. friction is low.  In this case, the variance of signed velocity is high.  In that case, we get some very high velocities compared to a spore which has a high drag coefficient gamma.

In October 2001, the labs directly observed high velocity movements of the spores under the microscope.  The spores flew off the slides.  This was observing the Brownian motion of the spores.  The original observations by Brown were on pollen.  Brown was a botanist.

The high velocity of the spores in October 2001 under the microscope was not due to em fields since it had no particular direction.  It could only be from Brownian motion.  Which means they directly observed the velocity distribution as high compared to normal spores they were familiar with.  This was equivalent to directly observing the drag coefficient as low.

Unless silicon in the coat causes this, there was an outer coating. In either case, the spores had a high velocity distribution from Brownian motion.  That is just fancy math talk for saying they dispersed.  The math of dispersion is the formulas of the velocity distribution under Brownian motion at the above link.

Since coated spores have a higher velocity then uncoated, over time, coated spores will suffer higher velocity collisions.  This will widen cracks and holes just like pot holes in a road.  Eventually, the coating is knocked off leaving the silicon inside the coat still there.  The uncoated particles have a high drag coefficient like normal spores and thus a low velocity distribution.

==Further reply to a comment by Dxer at Case Closed

High velocity follows from low drag coefficient. That is the formula.  We know the spores have velocity as seen under the microscope in October 2001 from Brownian motion because it was in all directions at once.

They observed a high velocity compared to normal spores.  That shows a low drag coefficient through the formula of Ornstein and Uhlenbeck for the variance of velocity in terms of the drag coefficient.  They are inverse.

Thus the drag coefficient was low for Senate anthrax compared to normal anthrax spores they had seen before under the microscope.  This was observed.

The drag coefficient being low means low friction.  A means for the drag coefficient to be low is an outer coating that doesn’t stick to whatever is around.  Thus the high velocity of the spores observed under the microscope compared to normal spores is evidence of an outer coating that reduced the drag coefficient.

The outer coating may or may not have been silicon although it seems likely.  The outer coating had to bind to something on the spore.  That could include the exosporium and the silicon in the coat.

Brownian motion of spores and the 1930 formula of Ornstein Uhlenbeck for the velocity distribution of spores in terms of a friction drag coefficient is  a basic part of spore science.  The energetic motion of the spores they saw under the microscope was Brownian motion exhibiting the OU formula for the velocity distribution.  Einstein won the Nobel for his simpler formula of Brownian motion.  Uhlenbeck won for his work with spin.

Having coated spores with a high velocity distribution from a low drag coefficient alongside spores that are uncoated with low velocity from a high drag coefficient will then be subject to the forces discussed which will tend to strip away the outside coating until there is equilibrium and all spores have high drag coefficient and low velocity distribution.

==

In October-November 2001 they saw under the microscope high velocity spores compared to normal spores.  The velocity came from Brownian motion in the Ornstein Uhlenbeck mode of analysis.  Thus they observed a low drag coefficient of the spores in October 2001.  Thus they observed something causing that low drag coefficient.  That would seem to imply a coating that lowered the drag coefficient.  So they observed a coating in October November 2001 appears to be what they observed from the high velocity.

==

The Ornstein Uhlenbeck velocity variance formula equates a high velocity to a low drag coefficient.  A low drag coefficient appears to best equate to a coating that lowers the drag coefficient.  Thus high velocity is equated to a coating.  Thus by observing the high velocity in October November 2001 under the microscope they observed a coating through the Ornstein Uhlenbeck math of Brownian motion.

The Sandia observations were also based on using formulas and indirect effects.  Thus Sandia observed no coating from its indirect analysis that came later and the microscopic examination of high velocity Brownian motion observed a coating in October November 2001.  The reconciliation is that the high velocity Brownian motion will eventually strip away the coating so that the spores are back to low velocity uncoated spores.  That is what Sandia saw.  So both sets of observations of the coating were indirect through math formulas.  They are reconciled through the high velocity Brownian motion itself stripping the coating over time to achieve equilibrium with low velocity uncoated spores.

Bruce Ivins Anthrax Vaccine Patent 6387665 shows couldn’t do it as FBI says

March 7, 2010

The FBI DOJ claim that Ivins produced the first batch of anthrax the weekend from Friday Sep 14 in the evening to Sunday evening Friday Sep 16 2001.  Ivins was in the lab 2 hours 15 minutes each night. Dxer at Case Closed Blog has found FBI 302’s that indicate this is the time it would take to check animals if Ivins was doing that work that weekend.  The second time period is from Friday Sep 28 2001 to Friday October 5 2001.  This period is when the FBI DOJ claim Ivins prepared the Senate anthrax letters in his lab at Ft. Detrick.  The following information shows that this would not be possible.  It would take 5 days using the single fermentor and that would produce too little anthrax.  The 5 days would require running during the week when others were present.  It would be a continuous 5 day run, not just at night for 5 days.  The following are Ivins own experiments using that fermentor reported in Ivins own patent application dated from 2000 before the 2001 time period.

Bruce Ivins et al applied for a patent in 2000 which was granted in 2002. This patent describes in detail the actual growing of anthrax by Bruce Ivins using the New Brunswick Bio-Flo 3000 that the FBI 302 report indicated was the fermentor at Ft. Detrick. The patent also describes use of the speed-vac. The patent gives a table with the yield in mg of protective antigen (PA) (not anthracis) using the 5 Liter fermentor after growth of several days.

http://www.freepatentsonline.com/6387665.html

“Fermentation conditions: The fermentations described here were carried out using a New Brunswick Bio-Flo 3000 equipped with a 5.0 liter working volume glass vessel and stainless steel headplate and hemispherical bottom cooling dish.”

“EXAMPLE 1

B. Anthracis ΔSterne-1(pPA102)CR4 was compared with its parent spore-forming strain B. anthracis ΔSterne-1(pPA102). Both organisms were plated onto sheep blood agar (a preferred medium for promoting bacterial spore production) and grown at 37° C. for 1 day, after which the temperature was lowered to 25° C. for 4 days. The two strains were also grown in liquid Leighton-Doi medium, which is designed to promote spore production, for 1 day at 37° C. followed by 4 days growth at 25° C. Growth from both agar and broth cultures were examined under phase contrast microscopy for the presence of spores. Growth from all four cultures were then resuspended in phosphate buffered saline to a concentration of about 10 9 colony-forming units (CFU) per ml. All four cultures were then heat shocked at 64° C. for 60 minutes to kill vegetative cells. Aliquots of 0.1 ml of the heat shocked material was then plated out onto sheep blood agar and incubated at 37° C. for 2 days. ”

“EXAMPLE 2

B. anthracis ΔSterne-1(pPA102)CR4 was grown in an FA medium fermentor culture. No spores were seen upon phase contract microscopic examination. Only medium-length and long chains of bacilli were seen. Dilution plate counts on the culture determined that the culture contained 1.86×10 9 CFU per ml. Three ml of culture was heat shocked at 60° C. for 60 minutes, then 0.2 ml was plated onto each of 5 plates of Tryptic soy agar. After incubation for 2 days at 37° C., no colonies were seen on the agar plates, indicating that spore production in the fermentor was less than 1 per 1.86×10 9 CFU. On two other fermentation runs with this strain, similar results were obtained. No revertants to the parent spore-forming phenotype were observed.

The above process using an FA medium fermentor culture was repeated using the parent strain B. anthracis ΔSterne-1(pPA102). Growth on the tryptic soy agar after heat shock resulted in a total of 1000 total colonies, indicating that the parent strain B . anthracis ΔSterne-1(pPA102) had about 1000 spores per ml in the FA medium, or 1 spore per 106 CFU in the non-heat shocked medium. ”

“TABLE 1
Summary of Aerobic ΔSterne-1(pPA102)CR4 Fermentations
Final
Final Final Yield Doubling
Conc. Yield (mg Specific Time
Fermentation (μg PA83/ (mg PA83/g Growth T D
Conditions ml PA83) DCW) Rate (min)
Aerobic, Batch 51 235 8.10 0.0132 min −1 53
Aerobic, Batch 64 301 10.7 0.0136 min −1 51
Aerobic, Batch 45 225 7.40 0.0136 min −1 51
pH constant
Aerobic, 68 360 ND 0.0116 min −1 60
Fed-Batch
(non-
continuous)
DCW = dry cell weight ”

The final protective antigen (PA)  yields ranged from 235mg to 360 mg. This was based on using the actual fermentor at Ft. Detrick in growth runs of 5 days as indicated in detail in the Example 1 quote.

You can search at the above link.

“For each O.D.600 determination, two appropriate dilutions were made and results were considered acceptable only when both dilutions yielded a linear response. DCWs were determined starting with a 2 hr point by centrifuging 10 mls of fermentation liquor at 11,953×g for 10 min, resuspending the cell pellet in 10 mls of sterile PBS and pelleting the cells again under the same conditions. The cell pellet was resuspended in a minimal volume of PBS and transferred quantitatively to a preweighted Eppendorf centrifuge tube and centrifuged at 14,000 rpm for 5 min. Excess PBS was removed and the cell pellet was dried in a speed-vac for 72 hrs under vacuum and a medium heat setting. A final analysis of the dry weight versus O.D. 600nm revealed that the relationship between the two parameters was adequately fit with a linear function. ”

The speed vac appears to be have been used for a small sample. This appears to be on a mLs sample, ie 10 milli liters.

This appears to settle it. Ivins could not produce the anthrax at Ft. Detrick using even the fermentor and the speed-vac. It would take 5 days.  (  The Senate anthrax contained 871mg per letter at least.  The yield of antigen is below this, but of anthrax spores might be higher than this amount.  Is it dried antigen?  A spore weighs less than a living cell so there is some offset depending on the weight of antigen in a living cell compared to how it was measured or estimated.)

The speed vac was used on a small sample of 10 milliliters. This is too small just as Ms. Ulrich indicated.

This patent was applied for in 2000. Ivins knew from this data that it would be impossible for him to produce the anthrax at his lab and convert it into high quality powder in the amounts in the Senate letters. The speed-vac could not process this volume. Ivins knew that. He is the principal person on the patent and is listed first out of alphabetical order. The patent also cites Ivins own papers.


Inventors:
Ivins, Bruce (Frederick, MD)
Worsham, Patricia (Jefferson, MD)
Friedlander, Arthur M. (Gaithersburg, MD)
Farchaus, Joseph W. (Frederick, MD)
Welkos, Susan L. (Frederick, MD) ”


Filing Date:
03/07/2000

“Method of making a vaccine for anthrax
United States Patent 6387665″

Search Detrick “bioflo 3000″

“Results 1 – 10 of about 56 for Detrick “bioflo 3000″. ”

This is further confirmation the New Brunswick 5-L Bioflo 3000 fermentor is the relevant fermentor at Ft. Detrick.

Search Bioflo 3000 Ivins

” Results 1 – 10 of about 20 for Bioflo 3000 Ivins.”

Mostly the patent but it appears another Ivins paper or two at least in the results.

This patent was previously discussed at Case Closed after Dxer posted an extract from it.

http://caseclosedbylewweinstein.wordpress.com/2009/09/25/nas-panel-meeting-92409-%E2%80%93-limited-information-available-since-the-nas-did-not-webcast-the-session-and-so-far-has-not-made-available-any-recording-of-the-session-or-powerpoints-of-the-pres/

The FBI 302 page 1 discusses the 5 Liter Bioflo 3000 as being at Ft. Detrick and then discusses a transfer to KBI.

http://foia.fbi.gov/amerithrax/847425.PDF

This is presumably the fermentor that was said by some to have been inoperative in fall 2001.

FBI misspells the name of the fermentor in the 302 report.

New Brunswick Scientific BioFlo III fermentor is correct name. Can search on Internet for it and find used models.

http://foia.fbi.gov/amerithrax/847425.PDF

See page 1 of 51 page pdf. FBI spells it:

“5 L Bio Flow III fermentor.”

Comment posted at

http://caseclosedbylewweinstein.wordpress.com/2010/03/06/anonymous-scientist-summarizes-the-fbis-case-against-dr-bruce-ivins-if-it-wasnt-so-serious-it-would-be-laughable/#comment-4355

==

You can see photos of the results of centrifugation by searching centrifuge pellet in Google and also in image search.

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

“Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate (“pellet”) to gather on the bottom of the tube. The remaining solution is properly called the “supernate” or “supernatant liquid”. The supernatant liquid is then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.”

You don’t end up with Senate letter quality dry non-clumping powder by just centrifuging. The pellet is still wet. Will dumping the pellet onto a plate and letting it air dry for 2 hours turn it into Senate letter quality powder?

Assuming the answer is no, is that why lyophilizers are used? But the lyophilizer was not available to Ivins and it would have required decontamination that would have been known to the lab. The FBI did not release any documents on the lyophilizer at Ft. Detrick? Nor mention it in the summary report that I recall. Nor documents on the speed vac such its size and capacity?

From page 19 of summary pdf

“Second, the evidence demonstrated that the perpetrator was familiar with key items of laboratory equipment used in microbiology research. All of the Ames anthrax existing in the 15
U.S. labs prior to the attacks was in liquid slurry form or on vegetative cell slants, rather than in powder form. Consequently, it was not possible for the perpetrator to merely steal an existing quantity of Ames spore powder “off the shelf,” because none was known to exist in the holdings of any laboratory. Even if the perpetrator stole a quantity of liquid Ames anthrax slurry, it would still have been necessary to dry the anthrax in order to produce a product like the one recovered from the envelopes. This drying procedure would have required either the type of laboratory equipment, such as a lyophilizer or speed-vac system, that was present in each of the 15 labs, or considerable time and space to air-dry. Alternatively, if the perpetrator stole only vegetative cells or a small quantity of spores to use as seed stock, not only would the perpetrator have to dry the anthrax, he would also have to subject the anthrax to two separate culturing and washing operations using an incubator and centrifuge.”

Footnote 17 on page 35 of summary

“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.”

These two quotations from the summary don’t seem fully consistent on the feasibility of centrifuging and air drying in 2 hours the quantities involved in the mailings.

If you air dry the paste, you spread it out with your fingers? It doesn’t clump when it dries when you do this?

If you can air dry 2 grams of powdered anthrax in 2 hours to get Senate letter quality, why do they use lyophilizers overnight?

Results 1 – 100 of about 22,500 for “lyophilized overnight”.

Meaning air dry the paste after centrifuging.

“leaving behind a pellet, or paste, capable of being dried in well under two hours.”

Does that mean air dried or does that mean dried with a speed vac or dried with a lyophilizer in 2 hours? Can you dry 2 grams of anthrax powder in 2 hours with a lyophilizer? Or is that overnight? Or more?
What about with the speed vac at Ft. Detrick?

Are there logs and sign outs for these devices? Have they been released for this time period?

“She said it would take about an hour to dry one milliliter of wet anthrax spores in one vial in a SpeedVac. It would have been impossible for Ivins to have dried more than a liter, which would have been required for the amount of anthrax sent in the letters, in the time frame they were mailed, Ulrich said. ”


Ulrich was a principal investigator in the diagnostic systems division at USAMRIID.

http://www.herald-mail.com/?cmd=displaystory&story_id=200518&format=html


She said Ivins was upset the FBI was watching him, but handled it as well as he could. “I’ve never even seen him angry,” Ulrich said.

“Ulrich said she worked with Ivins at the U.S. Army Medical Research Institute of Infectious Diseases in Frederick, Md., for about six years. The person she knew doesn’t match the troubled past Ivins is alleged to have had, she said. ”

Results 1 – 10 of about 19,100 for speedvac bacillus mL.

mL 1 milliliters = 0.0338140227 US fluid ounces

Results 1 – 10 of about 3,950 for speedvac bacillus uL.

uL is microLiter or 1/1000 of a mL.

These are typical experimental sizes for bacillus using a speedvac.

==

http://caseclosedbylewweinstein.wordpress.com/2010/02/28/new-york-times-fbis-anthrax-case-against-dr-ivins-is-not-convincing-independent-assessment-is-needed/

http://foia.fbi.gov/amerithrax/847425.PDF

page 44 interview of Ivins.

“RMR-1029, which is a large preparation of Bacillus anthracis Ames spoers compiled from multiple productions grown in both, shaker flasks at USAMRIID and fermentors at Dugway Proving Ground.”

So the Ba at USAMRIID was grown in the shakers which from a prior page in this same document, page 26, were at different places and only one shaker in any one place. These were hard to find space in according to the interview on page 26. Thus the type of run to produce 22 liters of anthrax used to help make RMR-1029 would be difficult to do. It would require many days on one shaker, if there is one in BSL-3. Ivins could not simply have put 11 flasks large enough to hold 2 liters of liquid and simply let them run together.

Ivins estimated 20 liters to make 2 grams of anthrax. The two Senate letters were at least 2 *.871 grams if no allowance for wastage is made. So given the set up at USAMRIID, Ivins time of 2.15 hours a night in BSL-3 would not give him the ability to have the shaker space and time needed for the runs for either weekend.

The FBI should have asked these questions and more. Instead, when an interviewee raises these points, the FBI agent notes them down but doesn’t follow up with questions related to them.

It appears the FBI agents think its someone else’s responsibility to figure out how it was done, not theirs individually or collectively. This appears to be part of an attitude of its not their job to think about the technical stuff.

When its not the FBI’s job to think about the technical stuff, then they are reduced to the gossip. When this is not enough, they try to lean on the person to get them to break. This pattern was repeated with Hatfill and then Ivins.

The FBI did not start out in 2001 with the question of how was this produced? Dxer mentions no 302’s from 2002. Is that because they asked the questions then and were told it required days and weeks with a good lab?

http://foia.fbi.gov/amerithrax/847425.PDF

Page 42.

“Blank recalls Ba samples being kept in the walk-in freezer for very short periods of time surrounding experiments. However these samples were usually already diluted for aerosolization. Blank believes that all glass impingers containing Ba may have been stored in this walk-in, but only for very short periods. Blank does not recall storage of any larger stocks (e.g. 500 ml flasks) of Ba.”

Ivins patent anthracis 6387665

http://www.google.com/#hl=en&q=ivins+patent+anthracis+6387665&aq=f&aqi=&aql=&oq=&fp=3d121c88310e67e3

Download Patent at Google

http://www.google.com/patents?vid=USPAT6387665

speed vac “two hours” overnight

http://www.google.com/#hl=en&ei=-CWUS7uPM8qm8AbcjOWoBQ&sa=X&oi=spell&resnum=0&ct=result&cd=1&ved=0CAUQBSgA&q=speed+vac+%22two+hours%22+overnight&spell=1&fp=3d121c88310e67e3

http://www.fbi.gov/anthrax/amerithraxlinks.htm

Detrick “bioflo 3000”

http://www.google.com/search?hl=en&q=Detrick+%22bioflo+3000%22&aq=f&aqi=&aql=&oq=

Results 110 of about 56 for Detrick “bioflo 3000”

Melanie Ulrich comments on Ivins

http://www.herald-mail.com/?cmd=displaystory&story_id=200518&format=html&autoreload=true

==

Comment submitted at:

http://anthraxvaccine.blogspot.com/2010/03/my-summary-of-unresolved-issues-in.html

==

The yields stated in the Ivins patent are for the yield of PA not of the anthrax itself.  (Jim White pointed this out on his blog.)

“protective antigen (PA) ”

“The data presented in Table 1 demonstrated that the PA yield on a unit volume and biomass basis,”

Claim one of the patent states

“1. A method of making a vaccine comprising: incorporating a protective antigen produced by recombinant asporogenic B. anthracis with a pharmaceutically acceptable carrier, wherein said recombinant asporogenic B. anthracis was isolated from a ΔSterne-1(pPA102) strain of bacteria and said recombinant asporogenic B. anthracis does not have the ability to bind a dye when grown on Congo Red Agar.

So the yield of PA understates the yield of the anthrax used to grow it.

The time of 5 days is the same however for the antigen and the anthrax bacillus it is in.  If we assume the antigen mass is a fixed ratio to the anthrax cell mass it is in, and that each anthrax cell has the same mass, then production of antigen mass is proportional to the production of anthrax cells.

The number of anthrax cells grown depends on the time period they are grown.  The mass of antigen follows the same path in time.  So if it takes 5 days to grow the antigens, then it takes 5 days to grow the cells.

(We can relax the assumption of fixed ratios made originally by allowing for fixed over time distributions of the ratios or cell size.)

A spore weighs less than a living cell so there is some offset  depending on the weight of antigen in a living cell compared to how it was measured or estimated.  So the antigen mass is less than a vegetative cell that contains it.  However, the spore weighs less than the vegetative cell.  How the antigen mass is determined matters too.  Is it converted to a dry powder and what loss of weight does that entail compared to a vegetative cell becoming a spore and then weighed as a dry powder of spores?

=Correction comments posted at Meryl Nass and Case Closed.

Final version at Meryl Nass  of one comment:

We must distinguish the vegetative cell, the antigen and a spore made from a vegetative cell.  A spore weighs less than a vegetative cell so there is some offset  depending on the weight of antigen in a vegetative cell compared to how it was measured or estimated.

The antigen mass is less than a vegetative cell that contains it.  However, the spore weighs less than the vegetative cell.  How the antigen mass is determined matters too.  Is it converted to a dry powder and what loss of weight does that entail compared to a vegetative cell becoming a spore and then weighed as a dry powder of spores?

FBI Anthrax report incorrectly reports 12 to 18 hour growth time anthrax

February 21, 2010

The FBI report on anthrax states that the protocol to grow anthrax is 12 to 18 hours.  See Footnote 25 page 40 of pdf which is numbered page 36 in the report linked to below.

http://www.justice.gov/amerithrax/docs/amx-investigative-summary.pdf

This is the protocol to grow micrograms or milligrams in lab runs for lab experiments.  To grow anthrax to produce grams of anthrax in letters takes 4 to 12 days using plates.  This is documented in the Carey et al paper linked to below.

==1

Production of Bacillus Spores as a Simulant for Biological Warfare Agents
Authors: Laurie F. Carey; Diane C. St. Amant; Mark A. Guelta; EDGEWOOD CHEMICAL BIOLOGICAL CENTER ABERDEEN PROVING GROUND MD

http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA426293&Location=U2&doc=GetTRDoc.pdf

They used a New Brunswick c25 floor shaker incubator for the runs using liquid as opposed to the NSM runs with plates.

See page 12 of pdf, 2.9.2.

This weighs 400 pounds.

Note they were doing 1 liter flasks. Those produce in the best runs somewhat under a gram. To produce 5 to 10 grams for the first letters would require 5 to 10 liters.

http://www.artisan-scientific.com/59721.htm

http://www.artisan-scientific.com/info/New_Brunswick_C25_Datasheet.pdf

See page 2 for photo. The unit is bigger than a man and weighs 400 pounds.

They started with 1ml of bacillus per liter of CD.

Since RMR-1029 was used as a source, they still had to grow it because the letters contained subtilis and silicon not in RMR1029.

Starting with more bacillus should shorten the production time, but it can not change the amount of growth media needed. The paper above also shows that yield is random and sometimes close to zero. It also shows that run time is 3 to 14 days with some runs duds at 14 days.

==2

They have another paper here:

http://www.dtic.mil/cgi-bin/GetTRDoc?Location=U2&doc=GetTRDoc.pdf&AD=ADA483822

AN IMPROVED METHOD FOR THE PRODUCTION OF Bacillus subtilis var. niger SPORES FOR
USE AS A SIMULANT FOR BIOLOGICAL WARFARE AGENTS- QUALITY ANALYSIS
Diane C. St. Amant, Laurie F. Carey, Mark A. Guelta
U.S. Army, Edgewood Chemical and Biological Command, APGEA, MD.

Please download them, you never know when they might not be there and at times they have been unavailable.

==3

Suppose your take 2 1 liter containers of growth media. Imagine its milk to seem concrete. Call the containers A and B. In A we put a single bacterium. In B we put two.

Suppose that a good run is 1 gram of spore equivalents, assuming a constant size of the spores. Suppose both containers have a good run. Then you end up with 1 gram of spores from each. But B will usually go faster by the time it takes a bacterium to split into two.

Note the container size is the same, 1 liter. The output is the same 1 gram. We get a yield of 1 gram per liter of growth media. This is true even though the starting number of bacteria is 2 times as many in case B as case A. Thus the final number of spores is a function of the growth media alone and not of the starting number of spores.

Final number of spores or grams (assuming a fixed size of spores) is a function of the liters of growth media (for the same growth media) and not of the starting number of spores.

==4

Another issue is how many grams of spores were in the letters. If one gram of spores is in a letter, and one liter of growth media is needed to grow one gram of spores (with a good run) then one gram of growth media is needed for that letter. If a spore powder is spores and only spores and is 1 gram, and if it takes one liter of growth media to make 1 gram of pure powder, then one needs one liter of growth media.

The Laurie F. Carey et al paper indicates the typical yield was more like .2 grams per liter in their experiments. (I am going by memory.)

If a letter had 1 gram of pure spore powder, and the yield was .2 grams per liter, one needs 5 liters per letter. 5 such letters needs 25 liters.

Ivins was shown using a glove box in a photo. That glove box didn’t look big enough for such a run. The paper used a floor shaker unit that weighed 400 pounds. If Ivins didn’t, his results would be worse, take longer and lower yield presumably.

==

Footnote 25 of FBI report page 36 at bottom of page, page 40 of pdf.

http://www.washingtonpost.com/wp-srv/politics/documents/amx-investigative-summary.pdf

http://www.justice.gov/amerithrax/docs/amx-investigative-summary.pdf

http://www.fbi.gov/anthrax/amerithraxlinks.htm

…Quote below

Each of these various devices plays a particular and critical role in the production of Ba spores. As a general matter, work on a pathogen of this type must be performed in a laboratory equipped with special safety devices and negative air pressure, so that any spills can be contained. With respect to spore production, generally speaking, a researcher would obtain a very small sample of spores from another source and transfer this sample to a growth medium, such as a Sheep’s Blood Agar (“SBA”) plate or a liquid preparation, that contained a nutrient source for the spores. An SBA plate could then be incubated to foster growth of spores. After an appropriate period of time, 12 to 18 hours was the standard protocol, the newly-grown spores would be harvested through an elaborate process, washed, and then spun down in a centrifuge to concentrate the spores. A fermentor can be used to grow large quantities of spores in a liquid preparation. Finally, as set forth supra in the Opportunity and Access section, a lyophilizer can be used to dry spores once they have been washed.

==

Compare FBI footnote to the report linked to below AN IMPROVED METHOD FOR THE PRODUCTION OF Bacillus subtilis var. niger SPORES FOR
USE AS A SIMULANT FOR BIOLOGICAL WARFARE AGENTS- QUALITY ANALYSIS
Diane C. St. Amant, Laurie F. Carey, Mark A. Guelta
U.S. Army, Edgewood Chemical and Biological Command, APGEA, MD.

http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA426293&Location=U2&doc=GetTRDoc.pdf

The pdf doesn’t let you cut and paste like the FBI one. So I will refer to page numbers of pdf, not of the report.

Read 2.9.1. page 12. This tells you that NSM refers to growth on plates.

Go to section 3.2 page 16 and read Tables 1 to 4. The growth times are 4 to 12 days for the NSM, which is the plates in their experiment.

Go to page 20, Table 6. This repeats the same information.

Go to 3.1.1 Page 15. It says NSM, plates, take 4 times longer than liquid media when including preparation and harvesting/processing time.

==

We appear to have a flat contradiction between the FBI report that says harvest plates in 12 to 18 hours and the subtilis growth paper that says it takes 4 to 12 days. So how do we reconcile these? Hypothesis: the answer is that 12 to 18 hours is a lab protocol to get micrograms or milligrams of spores or bacteria and the paper is about growing grams for sending in letters to kill people.

Suppose it takes 10 days for full growth. Suppose you start with 1 milligram equivalent of spores as live bacteria. You want 1 gram. So you want 1000. 2 to the 5 is 32, so 2 to the 10 is about 1000. So you need 10 doublings. (The rate of growth slows, but we ignore that.)

After 1 day, you have 2 milligrams. Labman can stop here. He only needs 2 milligram or less to do an experiment. Mailerman can not stop with 2 milligram. Mailerman instead has to continue the full 10 days. If mailerman stops at day 9, he has 1 doubling to go, so he has only 1/2 gram instead of the full gram. Mailerman has to continue the full 10 days.

The FBI was quoting Labman’s protocol not Mailerman’s protocol.

==

So why didn’t the FBI consult Laura Carey et al?  They had done the experiments and knew the numbers. Why didn’t the FBI ask them to write a section in the FBI report on the production of anthrax?  Why didn’t the FBI give the FBI report to Carey et al to check?

Hypothesis: In 2004, when this paper was released, the FBI and USAO DC intended that Carey et al would testify in United States v. Hatfill that Ivins and others in government labs could not have prepared and sent the anthrax because the growth times and large equipment required would have made detection very likely.   So in 2010, the FBI can’t have the scientists who did the experiments and know the numbers write the report or even review the report, because they had already committed to testify in 2004 for the USDOJ and FBI that Ivins could not have done it because of their experimental results.

There are no experimental papers for the FBI and USDOJ that support the 12 to 18 hour time for producing grams of spores to mail.  So they just make a bald statement of a protocol of 12 to 18 hours without explaining it.

At the same time, they reference a lyophilizer.  They knew the lyopholizer has to be cleaned when used to process anthrax and that this takes a day and requires the use of a room dedicated to that.  This came out from Ft. Detrick people on blogs.  The FBI knew that.  It was intentional deception to list the lyophilizer.

Its interesting to note Ivins comment about the lyophilizer course about how mathematical it is.  Ivins papers don’t contain equations and formulas.

==

anthrax equation author:Ivins

on Google scholar picks up 4 hits.

http://scholar.google.com/scholar?hl=en&q=anthrax+equation+author%3AIvins&btnG=Search&as_sdt=80000000000000&as_ylo=&as_vis=0

A logistic seems to be the extent of his math in papers and that is with coauthors.

==Info on C25 Floor Shaker used by Laura Carey et al

http://www.artisan-scientific.com/59721.htm

The C-25 Classic Incubator and C-25KC Classic Refrigerated Incubator Shakers are large capacity shakers utilizing an eccentric counter balanced drive to provide horizontal plane rotary motion in a 1″ (2.54 cm) circular orbit. A Proportional/Integral (PI) Microprocessor controller with instantaneous digital feedback controls the speed over a range of 50-400 rpm. The C-25 provides temperature control over a range of 7°C above ambient to 60°C, and the C-25KC provides temperature control over a range of 15°C below ambient (minimum 4°C ) to 60°C. Ambient temperature is defined as the temperature within one meter of the shaker. The shakers may be operated either continuously or in a timed mode via a programmable timer for shaking periods of 0.1 hr. to 99.9 hrs. For safe operation, both the C-25 and C-25KC are designed with a safety switch that automatically stops the shaker mechanism when the lid is lifted. In addition, the C-25 and C-25KC are equipped with visual and audible alarms that alert the user to the following conditions:
• The end of a timed run
• Deviations of shaking speed
• Deviations of temperature setpoint
• Power failure
• Lid open

That alarm would come in handy for late night lab users trying to get their work done, especially if it went off during a work day because they had to leave it running during the week.

Coworker: Why is the alarm going off on your 400 pound C-25 shaker incubator Bruce?

Bruce: I don’t know.

Coworker: Also why are using a big floor shaker incubator that doesn’t fit in your glove box?  We only do runs to produce micrograms around here.  Why do you need that giant heavy thing that doesn’t fit in your glovebox?

==

Note the C-25 is for liquid fermentation runs.  Without the 400 pound floor shaker incubator, the runs would take longer and/or have lower yields, or even be duds as sometimes happened even with the C-25, meaning no production after 14 days.

==Added comment at Weinstein blog to address question the thread was about.

Original comments from Jim White reposted at Case Closed

–Weinstein wrote:

Jim White believes a 100-fold math error in the Amerithrax investigation improperly excluded suspects

  • Substantial flaws still remain in the FBI’s explanation of the technical analysis on which they concluded that Bruce Ivins was the sole perpetrator of the anthrax attacks of 2001.
  • I have found what appears to be an error in the analysis of how much material from RMR-1029 would have been required to produce the spores used in the attack letters.
  • The result of this error is an overestimate, by a factor of 100, of how much material from RMR-1029 would have been needed to be used for each letter.
  • Partially because of this overestimate, the FBI excluded as suspects other researchers who received samples from RMR-1029, claiming that they lacked the expertise both to produce such a large volume of material and to then prepare it as attack material.

–Old Atlantic Answer:

Just to answer the original question.  The letter anthrax contained subtilis not in RMR-1029 and contained silicon not in RMR-1029.  That proves the letter anthrax was not directly from RMR-1029 but regrown.

The letters had 5+ grams in the first set and 2+ grams in the second set although there are those who sometimes argue over this.  The FBI should clarify what info it has.

Production of Bacillus Spores as a Simulant for Biological Warfare Agents
Authors: Laurie F. Carey; Diane C. St. Amant; Mark A. Guelta; EDGEWOOD CHEMICAL BIOLOGICAL CENTER ABERDEEN PROVING GROUND MD

These people actually grew grams and grams of subtilis spores.  They started out with empty plates and empty liter flasks, put growth media in them and put subtilis in them and actually grew bacteria and then turned them into spores and measured the weight they got.  They checked the growth media every 24 hours and did experiments every 24 hours on them to test how far along they were.

They used the New Brunswick C-25 floor shaker incubator unit to grow grams of subtilis in liquid media.  They used the necessary square feet of plates to grow on plates and get grams and grams of spores after centrifuging and drying.

This had to happen because the anthrax in the letters contained subtilis and silicon not in RMR-1029.

To get 5 grams of dry spores you have to start with a large amount of growth media, somewhere from 5 liters to 25 liters or even 50 liters.  Using twice as much bacteria to start can shorten the growth time by one generation’s growth time, but it doesn’t change the amount of growth media you need.  The amount of growth media, how many liters, is determined by how much bacteria you end up with not how much you start with.

The people who actually grew grams of bacteria report it took large scale equipment that doesn’t fit into Ivins’ glove box.  You can see the picture of the C-25 New Brunswick 400 pound floor shaker incubator.  Its bigger than a man.  Centrifuges to do the job are also large, I posted links to them at Meryl Nass in 2008.

It is not a matter of some notation in Ivins’ notebook suddenly changes everything.  The Laura Carey et al people actually grew the grams and grams of powder that are the equivalent of what is in the letters.  The second letters are reported as 99 percent pure in the recent FBI report.  So they are stuck with that.  Even one gram takes 3 to 14 days according to Carey et al, and that is using the C-25 400 pound floor incubator shaker and liquid media.

==

Originally posted at Weinstein Case Closed Anthrax blog

http://caseclosedbylewweinstein.wordpress.com/2010/02/20/jim-white-believes-a-100-fold-math-error-in-the-amerithrax-investigation-improperly-excluded-suspects-do-you-agree/#comment-3921

http://anthraxvaccine.blogspot.com/2010/02/fbi-case-closed-and-ivins-did-it.html

http://caseclosedbylewweinstein.wordpress.com/2010/02/20/meryl-nass-fbi-closed-the-case-before-the-nas-reported-its-findings-regarding-the-fbis-anthrax-science-why/

https://oldatlanticlighthouse.wordpress.com/category/anthrax-attacks/

==

The following links contain discussions of the growth times.

http://www.google.com/search?num=100&hl=en&safe=off&client=firefox-a&hs=oEr&rls=org.mozilla%3Aen-US%3Aofficial&channel=s&q=+dtic++site%3Aanthraxvaccine.blogspot.com&aq=f&aqi=&oq=

dtic  site:anthraxvaccine.blogspot.com

  1. Anthrax Vaccine — posts by Meryl Nass, M.D.: Additional comments

    http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA426293 Looks like the powers that be don’t want the people to read this paper.
    anthraxvaccine.blogspot.com/…/additional-comments-by-dr-popov-on.html – Cached

  2. Anthrax Vaccine — posts by Meryl Nass, M.D.: If the case is

    http://stinet.dtic.mil/cgi-bin/GetTRDoc?AD=ADA426293&Location=U2&doc=GetTRDoc.pdf showed that the level of Si is still only a fraction of 1%(estimated from
    anthraxvaccine.blogspot.com/…/if-case-is-solved-why-isnt-it-solved.html – Cached

  3. Anthrax Vaccine — posts by Meryl Nass, M.D.: FBI Proposal to NAS

    http://www.dtic.mil/srch/doc?collection=606943E40A9DF7C9&id=ADA426293 5. Was this paper intended to be delivered as evidence by the FBI in the prosecution
    anthraxvaccine.blogspot.com/2008/09/fbi-proposal-to-nas.html – Cached

  4. Anthrax Vaccine — posts by Meryl Nass, M.D.: Another anthrax

    http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA426293 PDF linking at this time. September 29, 2008 9:37 PM
    anthraxvaccine.blogspot.com/…/another-anthrax-accident-was-reported.html – Cached

  5. Anthrax Vaccine — posts by Meryl Nass, M.D.: Critique of the

    The dtic paper indicated multiple centrifuge runs of 30 minutes each. So if he had to use a 300ml capacity centrifuge he would have had to be in the lab far
    anthraxvaccine.blogspot.com/…/critique-of-chemical-signature.html – Cached

  6. Anthrax Vaccine — posts by Meryl Nass, M.D.: Trail of Odd Anthrax

    http://stinet.dtic.mil/cgi-bin/GetTRDoc?AD=ADA426293&Location=U2&doc=GetTRDoc.pdf. One can obtain a pretty good simulation of this EDX spectrum – note that
    anthraxvaccine.blogspot.com/…/trail-of-odd-anthrax-cells-led-fbi-to.html – Cached

  7. Anthrax Vaccine — posts by Meryl Nass, M.D.: Comments by

    http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA426293 I searched on: growing time bacillus spores.
    anthraxvaccine.blogspot.com/…/comments-by-professor-sergey-popov-on.html – Cached

Also

Popov  site:anthraxvaccine.blogspot.com

http://www.google.com/search?num=100&hl=en&safe=off&client=firefox-a&hs=lZW&rls=org.mozilla%3Aen-US%3Aofficial&channel=s&q=Popov++site%3Aanthraxvaccine.blogspot.com&aq=f&aqi=&oq=

  1. Anthrax Vaccine — posts by Meryl Nass, M.D.: Additional comments

    Additional comments by Dr. Popov on producing anthrax. 1. I agree with all scientific conclusions [of the Analytical Chemistry article] except for the one
    anthraxvaccine.blogspot.com/…/additional-comments-by-dr-popov-on.html – Cached

  2. Anthrax Vaccine — posts by Meryl Nass, M.D.: Comments by

    Sep 19, 2008 Dr. Popov worked in the former Soviet Union’s Biopreparat Program and is a professor at George Mason University. Having met with him several
    anthraxvaccine.blogspot.com/…/comments-by-professor-sergey-popov-on.html – Cached

  3. Anthrax Vaccine — posts by Meryl Nass, M.D.: Jahrling: Honest Mistake

    Sep 17, 2008 Additional comments by Dr. Popov on producing anth. Comments by Professor Sergei Popov on anthrax and .
    anthraxvaccine.blogspot.com/…/jahrling-honest-mistake.html – CachedSimilar

  4. Anthrax Vaccine — posts by Meryl Nass, M.D.: NYT: Portrait

    Jan 5, 2009 George Mason University professor and former Soviet bioweapons researcher Sergei Popov (who has also commented here), also distinguishes
    anthraxvaccine.blogspot.com/…/nyt-portrait-emerges-of-anthrax.html – CachedSimilar

  5. Anthrax Vaccine — posts by Meryl Nass, M.D.: September 2008

    Sep 1, 2008 Additional comments by Dr. Popov on producing anthrax …. Comments by Professor Sergei Popov on anthrax and on Dr. B.H. Rosenberg’s paper
    anthraxvaccine.blogspot.com/2008_09_01_archive.html – Cached

  6. Anthrax Vaccine — posts by Meryl Nass, M.D.: Anthrax Suspicions

    Additional comments by Dr. Popov on producing anth… Tracing KIller Spores: The science behind the Comments by Professor Sergei Popov on anthrax and .
    anthraxvaccine.blogspot.com/…/anthrax-suspicions-washington-post.html – Cached

  7. Anthrax Vaccine — posts by Meryl Nass, M.D.: Critique of the

    Dr. Popov responded that “This argument is purely hypothetical and implies a sophisticated generation of silica particles.” (Nano-scale powders were
    anthraxvaccine.blogspot.com/…/critique-of-chemical-signature.html – Cached

  8. Anthrax Vaccine — posts by Meryl Nass, M.D.: HOLT INTRODUCES

    Mar 3, 2009 It would be great to see someone like Serguei Popov leading the investigation into the science of the anthrax investigation.
    anthraxvaccine.blogspot.com/…/holt-introduces-anthrax-commission.html – Cached

==

dtic site:caseclosedbylewweinstein.wordpress.com

http://www.google.com/search?num=100&hl=en&safe=off&client=firefox-a&hs=fcW&rls=org.mozilla%3Aen-US%3Aofficial&channel=s&q=dtic+site%3Acaseclosedbylewweinstein.wordpress.com&btnG=Search&aq=f&aqi=&oq=

Popov site:caseclosedbylewweinstein.wordpress.com

http://www.google.com/search?num=100&hl=en&safe=off&client=firefox-a&hs=LdW&rls=org.mozilla%3Aen-US%3Aofficial&channel=s&q=Popov+site%3Acaseclosedbylewweinstein.wordpress.com&btnG=Search&aq=f&aqi=&oq=

==

dtic site:oldatlanticlighthouse.wordpress.com

http://www.google.com/search?num=100&hl=en&safe=off&client=firefox-a&hs=SJr&rls=org.mozilla%3Aen-US%3Aofficial&channel=s&q=dtic+site%3Aoldatlanticlighthouse.wordpress.com&aq=f&aqi=&oq=

  1. Vahid Majidi « Old Atlantic Lighthouse

    The dtic paper did 30 minute centrifuge runs and did multiple ones. So for 5 liters, which is the minimum size run he was doing, it would take 6.5 hours.
    oldatlanticlighthouse.wordpress.com/category/vahid-majidi/ – CachedSimilar

  2. Anthrax attacks « Old Atlantic Lighthouse

    The dtic paper did 30 minute centrifuge runs and did multiple ones. So for 5 liters, which is the minimum size run he was doing, it would take 6.5 hours.
    oldatlanticlighthouse.wordpress.com/category/anthrax-attacks/ – Cached

  3. 2009 February « Old Atlantic Lighthouse

    Note the paper I cite above from dtic, if you estimate that for each liter This shows the dtic paper and the Bacchus project results are consistent and
    oldatlanticlighthouse.wordpress.com/2009/02/ – Cached

The FBI footnote 25 on page 36 of document, page 40 of pdf, speaks of a standard protocol to harvest in 12 to 18 hours.

After an appropriate period of time, 12 to 18 hours was the standard protocol, the newly-grown spores would be harvested through an elaborate process, washed, and then spun down in a centrifuge to concentrate the spores.

Standard protocol for what? For producing grams of anthrax to mail to victims?  If you are producing kilograms for military scale use, you use large fermenters as in the Soviet Union facilities.  If you are producing for lab experiments, you don’t produce 5 liters of liquid material or the equivalent on plates.  You produce a tiny amount for use.

Dr. Popov in his initial thinking at Meryl Nass was thinking of what would be quite small.  That is the normal lab thinking.  Which is not to produce grams to send out in letters to kill people.  The standard protocol is to produce a few small plates, enough to go in a test tube or on a plate under a microscope.  They don’t make liters of the stuff for a lab experiment.

So the standard protocol phrase gives it away.  Its not the standard protocol to produce grams of anthrax powder, its the standard protocol to produce micrograms or milligrams on plates for use in a lab experiment.

Why didn’t the FBI have Laura Carey et al read their report?  Why didn’t they write the section and do the numbers?  Because they had already agreed to testify in 2004 for the FBI and USDOJ that based on their paper and their analysis of the numbers, it would have taken days using proper equipment to grow the 5+ grams of anthrax in the first batch of letters and 2+ grams in the second set and that their results exclude Bruce Ivins specifically and all others like him.

So Laura Carey et al couldn’t come back in 2010 to prepare or even review a report claiming Ivins did it in a weekend in September and then over a week in October but at night when he couldn’t do continuous runs during the week using a New Brunswick C-25 400 pound incubator shaker as Laura Carey et al did.  These are people with names.  Let the journalists call them up and ask them.  If they say they can’t comment, report that.

==

The 1 volume 888 page Warren Commission report contained an appendix reviewing alternative theories and giving reasons against them.  This was not in the FBI report in 2010, 9 years after the 2001 attacks, despite blogs being full of them.  Including blogs focused on them with experts commenting such as Meryl Nass and Weinstein’s Case Closed.

http://www.archives.gov/research/jfk/warren-commission-report/appendix-12.html

http://www.archives.gov/research/jfk/warren-commission-report/

Volume 1 was printed in 1964, 1 year after the Kennedy assassination.  That was in the days of typewriters.  The FBI only had to cut and paste from blogs and then give their reasons the alternative theory is wrong.  They didn’t do that.  Even Ed Lake talks about alternative theories.

http://www.anthraxinvestigation.com/

The FBI doesn’t cite this Laura Carey et al paper that likely they commissioned for use at trial against Hatfill to exclude Ivins specifically by name.  Its time that is tracked down and put in the public record.  Isn’t that what they give Pulitzer Prizes for?  Or used to?  Isn’t that 19th century Nellie Bly journalism from the Golden Days of Investigative Reporting.  Did that end with Woodward and Bernstein?

==

The Laura Carey et al paper is a great scientific paper.  It is accessible to the lay reader.  But it requires work and you may have to Google some scientific terms.  Those have simple explanations and definitions.   Skip around in the paper don’t try to read it from start to finish.

This paper requires many rereadings to get most of it.  Some of the conclusions were held back.  Its implicit that they were surprised how variable the results were.  Ordinary lab experience of the type the FBI talked about, 12 to 18 hours, is evidently not that sensitive to the amount grown, whereas for letters there is big difference between 1 gram and .1 gram.  Every rereading you learn more, but you learn from the first one.  This is the mark of a great scientific paper which this is.

Battelle wins Nobel Prize in Medicine for anthrax science

October 5, 2009

(Spoof) Battelle Labs finally won the Nobel Prize in Medicine for its role in the anthrax attacks and their coverup.

To Battelle Labs: For autoclaving anthrax from the letters and then saying it was puppy chow.

Also mentioned

Vahid Majidi, head of FBI WMD

Jeff Taylor, US Attorney DC appointed without Senate approval, first one ever.

Dr. James Burans, Battelle, DHS and the bioweapons industrial complex aka National Bioforensic Analysis Center (NBFAC).

Dr. James Burans

Battelle autoclave anthrax

Anthrax Atrocities

the anthrax spores before studying them, so Battelle processed its sample in an autoclave and got a lower value for virulence and dispersal qualities.
http://www.prisonplanet.com/did_the_government_ok_the_anthrax_attacks.htm – Similar

Salon.com Mobile – CargoCult

Aug 5, 2008 That is the very same Battelle who operated the Dugway Utah you stuck your anthrax in an autoclave, and you turned it into hockey pucks.
letters.mobile.salon.com/…/author/index13.html – CachedSimilar

http://www.homeland1.com/homeland-security-products/air-quality-testing-gas-detection/articles/449338-security-giant-fbi-to-launch-massive-biodefense-center-in-md/

Anthrax attacks do pay.

http://letters.mobile.salon.com/opinion/greenwald/2008/08/18/anthrax/view/index20.html

http://www.fredericknewspost.com/sections/news/display.htm?storyID=78992

Dr. James Burans, associate lab director of the Department of Homeland Security’s National Biodefense Forensic Analysis Center, said the anthrax powder used in the mailings could have been produced by one person in three to seven days.

Evening Friday Sep 14 2001 to Evening Sunday Sep 16 2001 is only 48 hours.  Moreover, Ivins was only in the lab 2 hours 15 minutes each evening.  Could you do it with those restrictions?  Not really.

Burans Battelle employee

http://anthraxvaccine.blogspot.com/2008/09/tracing-killer-spores.html

http://anthraxvaccine.blogspot.com/2009/01/more-pictures-complete-exosporium.html

Case reviewed here including Burans Battelle relationship:

http://caseclosedbylewweinstein.wordpress.com/2009/10/04/barry-kissins-report-to-congressman-rush-holt-the-truth-about-the-anthrax-attacks-and-its-cover-up/

http://caseclosedbylewweinstein.wordpress.com/2009/10/05/robert-stevens-died-eight-years-ago-today-the-first-victim-of-the-still-unsolved-anthrax-attacks/

Actual Nobel Prize Medicine

http://news.yahoo.com/s/ap/20091005/ap_on_re_eu/eu_nobel_medicine;_ylt=AotnIbs9aQpi0Or3g3QGaUes0NUE;_ylu=X3oDMTJlMDEzbmtrBGFzc2V0A2FwLzIwMDkxMDA1L2V1X25vYmVsX21lZGljaW5lBHBvcwM2BHNlYwN5bl9tb3N0X3BvcHVsYXIEc2xrAzNhbWVyaWNhbnNzaA–

http://nobelprize.org/nobel_prizes/medicine/laureates/2009/index.html

http://nobelprize.org/nobel_prizes/medicine/laureates/2009/press.html

Press Release

5 October 2009

The Nobel Assembly at Karolinska Institutet has today decided to award
The Nobel Prize in Physiology or Medicine 2009 jointly to

Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak

for the discovery of

“how chromosomes are protected by telomeres and the enzyme telomerase

Summary

This year’s Nobel Prize in Physiology or Medicine is awarded to three scientists who have solved a major problem in biology: how the chromosomes can be copied in a complete way during cell divisions and how they are protected against degradation. The Nobel Laureates have shown that the solution is to be found in the ends of the chromosomes – the telomeres – and in an enzyme that forms them – telomerase.

A possible partial solution to the anthrax mystery

January 21, 2009

https://www.blogger.com/comment.g?blogID=6592607595936297457&postID=2951234322664707881&page=1&isPopup=true

http://anthraxvaccine.blogspot.com/2009/01/critique-of-chemical-signature.html

A possible solution to the Bruce Ivins FBI anthrax mystery is outlined in a comment at Meryl Nass produced below.  This arose from an extended discussion of many but including Ed Lake and Attorney Barry Kissin, Ross Getman, some anonymous experts, and others.  Perhaps Vahid Majidi and Director Mueller of the FBI could consider this.

Article by Paul de Armond linked to by Ed Lake :

http://www.albionmonitor.com/0208a/anthrax.html

Ed, thanks for the link and discussion.  Of course, if you take everything in the article linked to as valid, it would tend away from Ivins in my view.  But I wouldn’t hold anyone to such a standard.

http://en.wikipedia.org/wiki/Endospore#Formation_and_destruction


Reactivation of the endospore occurs when conditions are more favourable and involves activation, germination, and outgrowth. Even if an endospore is located in plentiful nutrients, it may fail to germinate unless activation has taken place. This may be triggered by heating the endospore. Germination involves the dormant endospore starting metabolic activity and thus breaking hibernation. It is commonly characterised by rupture or absorption of the spore coat, swelling of the endospore, an increase in metabolic activity, and loss of resistance to environmental stress. Outgrowth follows germination and involves the core of the endospore manufacturing new chemical components and exiting the old spore coat to develop into a fully functional vegetative bacterial cell, which can divide to produce more cells.

One article I saw in a search talked about 3 to 7 hours for one stage of this or maybe all 3.  But some said it can be up to 60 days or never.  The article I linked to above on runs found some dud runs. Even your linked article said it looked like multiple runs some duds were tried.


When a bacterium detects environmental conditions are becoming unfavourable it may start the process of sporulation, which takes about eight hours.

If the idea is to start Friday night and have spores ready Sunday morning, he is going to take 8 hours or more to activate to ready for exponential growth.  But then its almost time to stop and go to work to put them to sleep, sporulation.

Ivins needed to be done Sunday morning to prepare the envelopes and clean everything up and get out.

Ivins knew the above biology already.  He knew that starting with spores Friday night and ending with spores Sunday morning meant the time to grow would be zero.  Moreover, he knew from long experience the time to activate was highly random and the initial growth highly random.  The dud runs in the article I link to above show that.

The evidence from the article linked (by Ed) is that 10 percent of some of the first letters were spores and the rest growth material.  Ivins knew that is what would happen.  Ivins knew his schedule, and he knew spores to growing dividing cells back to spores by Sunday morning was a fool’s errand.  So why did he do it?  Dr. Popov in his discussion indicates it takes days to do anything meaningful including prep time.  (There is also thorough cleaning to avoid detection here.)

Alternative scenario.

Someone someplace else as part of a conspiracy started growing spores the week after 9/11 on orders from someone.  They were told to grow and prepare them.

Then the order came in to take what they had and mail them right away.  That is, it was unexpected to stop the growth and mail what they had.  That means it wasn’t Ivins. Ivins would have known he had to be back to spores Sunday morning.

So someone else in place X was told to prepare a lot of anthrax by growth.  But then within a couple of days of that order they were told to stop and do sporulation and mail out what they had.  This means it wasn’t Ivins acting alone.

Moreover, the place was not Ivins lab, it was someplace they expected to be able to run a large fermenter or shaker incubator for 7 to 14 days like in the paper I linked to.  Run to completion and test for completion like the paper did.  But then they were surprised and had to sporulate what they had and mail it. That means it wasn’t Ivins.

Its a classic detective story plot.  (Too bad the FBI can’t figure these out isn’t it? But that is for the amateur detective?) We just needed to activate the little gray cells together mon ami.  N’est-ce pas?

==

Searches that are interesting are

bacillus sporulation

and activation, germination, out-growth, anthracis, subtilis, etc.

==Prior posts

Old Atlantic Lighthouse said…

I would like to bring up the government’s own tests on how long it takes to incubate that they released in 2004 when they wanted to prove Ivins and others like him couldn’t do it in a prosecution of Hatfill.

http://oai.dtic.mil/oai/oai?&verb=getRecord&metadataPrefix=html&identifier=ADA426293

http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA426293&Location=U2&doc=GetTRDoc.pdf

They used a

New Brunswick c25 floor shaker incubator

See page 12 of pdf, 2.9.2.

This weighs 400 pounds.

Note they were doing 1 liter flasks. Those produce in the best runs somewhat under a gram. To produce 5 to 10 grams for the first letters would require 5 to 10 liters.

http://www.artisan-scientific.com/59721.htm

http://www.artisan-scientific.com/info/New_Brunswick_C25_Datasheet.pdf

See page 2 for photo. The unit is bigger than a man and weighs 400 pounds.

There is a smaller unit

http://www.artisan-scientific.com/57635.htm

It weights 230 pounds.

http://www.artisan-scientific.com/info/Innova_4230_Datasheet.pdf

The paper above ran at 30 deg C using the incubator shaker. So if one didn’t use something that good it would take longer.

They started with 1ml of bacillus per liter of CD. So that is a factor of 1000, which is roughly 2 to the 10 power. So if you start with more you cut down the growth time needed. Even so, there doesn’t appear enough time.

Look at Table 6 on page 20 of the pdf.

They checked the runs every 24 hours to see how far along they were. The best run was 3 days to produce .5238 grams for the starting liter of CD. So to get 5 grams you need 10 liters of CD. Note even if you start with more than 1 ml of bacillus you still need the 10 liters of CD to get the 5 grams.

If we take 10 generations as 3 days, we get approximately 3 generations in 1 day, i.e. a factor of 8. Divide the 5 grams by 8 and we get 5/8 grams he needed to start with to get done in 24 hours. If he had a liter flask and the ratios are the same, then he needed to use 5/8 of his reference flask, if it was full to 1 liter.

Note, he could have put some back at the end.

If things didn’t go so well as this perfect run, it can’t work.

He also has to do centrifuging, rinsing, re-centrifuge, and eventually lyophilize.

Every piece of equipment he used to make it go faster had to be cleaned and put back where it belongs.

To do it all in one run and get the best possible growth times he had to have the best equipment.

Note if he had the 400 pound floor incubator, then if anthrax got on the bottom of it he couldn’t clean it. Even the 230 pound unit, he couldn’t clean.

Did the BSL3 have these big units?

As you consider all the other steps needed, including cleanup, intermediate steps, getting the CD ready, lyophilizing, putting it in letters, covering his tracks, etc. it becomes hard to believe he did it from Friday night to Sunday the first weekend.

Table 6 in the paper records total dud runs. Some runs take 7 days. For those runs even if he started with all of the RMR 1029 flask and ran 1 day, he wouldn’t get 5 grams.

Because at 7 days, you are doubling at 7/10 of a day. So if you double twice your at most 1 gram you get 4 grams. You then have to put 1 gram back, so you have 3 grams.

The paper above is sort of difficult to read and get everything out you need at one sitting. Its important to go over it more than once. That’s the benchmark.

To run 10 liters, Ivins needed a big C25 style. There are other fermenter units not as good. But they likely will take longer. It still takes at least a 10 liter one. If he didn’t get a perfect run, we should think 25 to 50 liter fermenter.

If you search on

“50-liter fermenter” anthrax

You get some interesting hits. That is what they had at Dugway in Utah for Project Bacchus.

http://www.pbs.org/wgbh/nova/transcripts/2815bioterror.html

January 19, 2009 10:05 AM

===

Mr. Lake is mistaken.

Gerry Andrews, Ivins’ supervisor between 2000 and 2003, is not under a gag order and has addressed all these issues as quoted above and elsewhere.

Two other supervisors Jeffrey Adamovicz and Dr. W. Russell Byrne, also disagree and have spoken publicly on these issues. Jeffrey Adamovicz worked with him for more than 12 years. Dr. A was his boss from 2003 to 2004. He remembers the day the scientists opened that envelope, placed in a double-sealed bag inside a protective hood designed to deal with dangerous pathogens. “The anthrax was floating around inside the bag,” Mr. Adamovicz said. “It was very scary.” He said he turned to Dr. Ivins and said, “That stuff is amazing.” “Yes, it is unbelievable,” he recalled Dr. Ivins replying. “I have never seen anything like that.”

Dr. Ivins, his colleagues said, argued that al Qaeda was responsible. “He was very passionate about this,” former boss Jeffrey Adamovicz said.

None of these men are under a gag order contrary to what Mr. Lake suggests. Mr. Lake apparently has never contacted them to be apprised of the facts. He instead offers his own opinion even though he is not even a microbiologist.

January 20, 2009 6:37 PM

Blogger Old Atlantic Lighthouse said…
Ed, there was some prior discussion on the number of square feet of plates it would take.

http://anthraxvaccine.blogspot.com/2008/09/comments-by-professor-sergey-popov-on.html

Based on the paper cited above,

http://oai.dtic.mil/oai/oai?&verb=getRecord&metadataPrefix=html&identifier=ADA426293

http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA426293&Location=U2&doc=GetTRDoc.pdf

I came to some estimates of square feet of plates from 15 square feet on up. The paper did runs with plates and you can get some numbers there and in the prior posts on this subject.

http://anthraxvaccine.blogspot.com/2008/09/additional-comments-by-dr-popov-on.html

Popov estimates at least 100 plates per letter.

Plates take up too much space and in these volumes are a lot of work. Plus you have to have that many plates. It then takes a long time to work the plates at each stage.

There is also the activation time or dormancy time or time to first generation. I have searched on various terms. There are many papers on this, some sponsored by the FBI. The following search gets many interesting hits.

bacillus spores “time to germinate”

One can substitute or try phrases like anthracis, “germination time”, dormancy, endospore dormancy
“spores remained dormant”

and so on. The articles I found didn’t seem to hit spot on the issue of how long until exponential growth starts from the time you take spores and put them in the growth medium.

If we go back to the paper I linked to further above that did the trials of growth times, some runs were duds. It may be that it takes hours or even days to start.

Also as the container is saturated, it should slow. Hypothesis:

dN = k N(Nmax – N) dt

Nmax is the maximum number of spores the growth medium in a flask can support. If he spiked a flask with more starting spores, then he will have a slower growth rate than the average from the paper I cited which started with 1 ml of spore solution per liter of growth medium.

January 20, 2009 9:05 PM

Delete

Blogger Old Atlantic Lighthouse said…
Ed, your point about the 10 percent or so of the first letters being actual spores is important. I was thinking this might be an issue, but didn’t have time to research it or factor it in. I believe Dr. Popov mentioned something in his discussion, but I am not sure if he had a specific number. Can you document the source on the 10 percent figure?

I admire your taking on all comers in a spirited debate. We can learn a great deal from such a discussion and learning science this way is in general valuable.

January 20, 2009 9:39 PM

==Rougher notes which may have repetition of the above

http://www.ncbi.nlm.nih.gov/pubmed/18984151

http://www3.interscience.wiley.com/journal/120832256/abstract?CRETRY=1&SRETRY=0

http://www3.interscience.wiley.com/journal/120832256/abstract


Early administration of antibiotics after
exposure is essential – with inhaled anthrax, a delay of just a
few hours is enough to significantly reduce the chances of
survival. Because spores can take a long time to germinate,
antibiotic treatment should continue for 60 days.
Source: JAMA, 1999. 281, 1735-1745.

http://www.parliament.uk/post/pn166.pdf

bacillus spores “time to germinate”

http://www.sciencedaily.com/releases/2007/06/070604164928.htm


The research is funded by LLNL’s Laboratory Directed Research and Development program, the Defense Advanced Research Project Agency (DARPA) and the Federal Bureau of Investigation.

https://www-pls.llnl.gov/?url=about_pls-scientific_staff-malkin_a

https://www-pls.llnl.gov/?url=about_pls-scientific_staff-malkin_a

http://www.pnas.org/content/104/23/9644.full.pdf


Spore and Vegetative Cell Preparation: Purification and Germination
Conditions. B. atrophaeus (ATCC 9372) was obtained from the
American Type Culture Collection (ATCC, Manassas, VA).
Spores were produced by using Schaeffer’s sporulation medium
and purified as described (36). Spore germination was
induced by the addition of 100 mM L-alanine, 1.65 mM
L-asparagine, 2.8 mM D-glucose, 2.8 mM D-fructose, 5 mM
potassium chloride, and 25 mM TrisHCl buffer, pH 8.0. The
germination time course was characterized by phase contrast
microscopy before performing AFM imaging. More than 95%
of spores turned phase dark with

http://www.pnas.org/content/104/23/9644.full.pdf

page 5

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1877984

http://www.ncbi.nlm.nih.gov/pubmed/17007517


The time-to-germination (t(germ)), defined as the time required for the CaDPA band intensity to decrease to the midpoint from its initial value, was found to be stochastic for individual spores with a typical value of approximately 30 min under the experimental conditions.

http://www.ncbi.nlm.nih.gov/pubmed/17007517

bacillus “time-to-germination”

http://mic.sgmjournals.org/cgi/content/full/148/7/2089

http://www.ncbi.nlm.nih.gov/pubmed/11964118?dopt=Abstract

bacillus spore “germination time”

activation

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1539616

http://bodyandhealth.canada.com/channel_section_details.asp?text_id=1484&channel_id=1020&relation_id=10875

endospore dormancy

“spores remained dormant”

http://www3.interscience.wiley.com/journal/119420121/abstract

==

Ed, there was some prior discussion on the number of square feet of plates it would take.

http://anthraxvaccine.blogspot.com/2008/09/comments-by-professor-sergey-popov-on.html

Based on the paper cited above,

http://oai.dtic.mil/oai/oai?&verb=getRecord&metadataPrefix=html&identifier=ADA426293

http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA426293&Location=U2&doc=GetTRDoc.pdf

==

The actual spores in the first letters may have been mostly grown as opposed to mostly started with.  That means the start time was several days earlier.  If it was in the middle of a run that takes 7 to 14 days it might have started from before to after 9/11.

==

The FBI in its released warrants or affidavits or its news conferences made it seem like the time he spent in the BSL3 was the conclusive proof he did it. Daschle said so and the FBI briefed him special.  The FBI didn’t say it had secret evidence of his guilt and that the time in the BSL3 was not a major part of the evidence.

According to the FBI, Ivins was in the BSL3 2 hours 15 minutes each day Friday, Sat, Sun after 9/11.  That is the amount of time he would have had.  It may be that he was in and out.  Could he go from spores to cells back to spores to envelopes and clean everything and dismantle all makeshift equipment in that time?

If he had 5 liters of solution
on Saturday, how could he reduce it in time to powder spores?  (The evidence from above is that there was an attempt to grow a lot.  Note there were 5 letters in the first mailing according to current thinking according to the Wiki article.)

1) Build his own lyophilizer and dismantle it when finished.

2) Use a large capacity lyophilizer at the lab.  (This seems out.)

3) Spray dryer.  Likely not available.

4) Speed vac.  Probably too small and might run too long?

Think about boiling a one quart pot of water on the stove until its dry.  How long does that take?  Note that boiling water with anthrax for over 30 minutes will likely kill it all according to sources that I found.  Heating to 40 deg C without a speed vac or hood or special equipment to remove the vapor would likely take too long.  There is always the contamination issue of the lab as well to consider.

He would presumably centrifuge the solution first, if he had sufficient centrifuge capacity.  He then has to clean the centrifuge.  All the equipment he uses has to be cleaned to avoid detection.  He has to wear his suit while doing cleaning and every other step.  That makes it take longer.

Does it make sense to try to make 5 grams of anthrax starting with spores in 36 hours with a limit of 2 hours 15 minutes per day in the lab?

==

Let’s recap two competing scenarios, among more I know.

In scenario one, a lab with all the equipment to do 5 to 10 gram powder runs is set up somewhere.  Workers can work without having to worry about being discovered.  They have the top equipment of every type, large floor shaker incubator, big centrifuges, spray dryers and lyophilizers, test equipment, etc.

This lab is given the order to produce 5 to 10 grams.  Its sets that up and starts running.  But the other comes in to stop and hand over what they have to someone else.

Then in the second batch, they are given the order to prepare more.  Possibly right after this aborted attempt.  They just go back to work immediately.

Note that the second letters were mailed Oct 19.  If you think about a 14 day run in the paper I linked to above and then days to process the powder, it could take 3 weeks going flat out to produce good powder for the next run.

In the Ivins case, Ivins didn’t have the equipment the first time, nor the second.  Ivins never had the good setup described above or in the paper I linked to previously.  In the Ivins scenario, he doesn’t start again until 2 weeks later and has only 1 week to do it. So again, he has too little time, can only work part time at it, and doesn’t have the equipment.

In comparing likelihoods, each run is less likely for Ivins.  So the total likelihood of Ivins is much lower looked at from this point of view.

==

The reason Friday Sep 14 to Sep 16 is so important is that the FBI presented this as the psychological proof Ivins did it.  Daschle was briefed by the FBI and he said he believed because of the nights before the mailing.

But now we are told it wasn’t even possible to prepare the anthrax in the nights before the first mailing.  This is presented as if the FBI had never said or implied that.   Did they go back and tell Daschle this?

The reason Friday to Sunday is so important is the chance not to be seen.  If it takes 3 days (72 hours) or more to let an incubator or fermenter run in the lab, then it has to run during a weekday, Friday or Monday and possibly more.

That means others can see it.  We are talking about preparing 5 grams or more in a run.  That is a bigger run of anthrax than has been done at Ft. Detrick in the recent era.  So it would be noticed.  It would be a giant run relative to normal.

Moreover, the stats on the paper I showed show that to get 5 grams of powder one should really aim for more like 25 liters to 50 liters because its unlikely that a liter will produce a gram of powder.

The strongest evidence connecting Ivins to the mailings is the lab time.  That’s the FBI crown jewel of evidence.  But if in fact, he would have to let a large run of 5 liters or more in a flask run (or hundreds of plates) then it is not such great evidence because it would have been noticed.

Note also for 5 grams using Dr. Popov’s 100 plates per letter estimate we get 500 plates.  1 minute per plate comes to 500 minutes, longer than a 2 hour 15 minute period.  So he didn’t have time to manipulate 100’s of plates in each step, set up, drying and scraping.  Remember, you wanted to have the plates scraped.  Well scraping 500 plates takes more than 2 hours 15 minutes.

That the first letters show an attempt to grow shows he needed to.  That means the anthrax of the second letters had to be grown.  But that means running on week days.

The first letters were unsatisfactory.  So new anthrax had to be grown.  If it takes 72 hours, then he had to run on a weekday, either with 200 or more plates for 2 grams (if not 400 for 4 grams) or 2 to 20 liters of material in a rotating shaking fermenter.  If he used a flask only, his runtime would be longer.

To get 2 grams of powder would likely take 7 days to 14 days not 3.  Then it has to be dried.

Air drying will take time.  He doesn’t have that on the first weekend.  If he air dries he has to stop on Saturday.  In which case, how did he expect to get 5 grams to grow if he was going to stop on Saturday to air dry?

Some Ivins emails have been released.  Some contain extensive statistical output before 9/11/2001.  So he knew how to count.  They have tests of statistical significance.  So he understood variability.  The paper I cited before shows many runs are duds.  Ivins knew that.

The FBI used the times in the lab before the mailing as its proof for Daschle.  But those times are not adequate to do all the steps.  So the FBI case is gone.  What is the evidence left?

==

Ed, try commenting on following.

http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA426293&Location=U2&doc=GetTRDoc.pdf

Page 12 of pdf methodology.  Page 20 table of results.

Note that both plates (NSM) and liquid used an incubator.  In each case, 3 days was the best time.  But some runs were duds even after 10 to 14 days.

The person thought they could grow at least 5 grams it appears, but stopped with only .5 grams for the first letters, relying on Ed’s linked source and a guess of 5 grams material.  But maybe its 10 grams and 1 gram?

It appears someone expected to run 3 to 14 days and harvest a full set.  Then they had to stop and give what they had to someone or use it themselves.  Then immediately after that, i.e. around Sep 18 they started on the run for the second set.

Did Ivins have a centrifuge to handle 5 liters or more?  The paper talks about centrifuging multiple times, each time taking 30 minutes.  If Ivins had 2 hours 15 minutes max on each lab day in the first set, he doesn’t have time to centrifuge the 5 liters even once if he was limited to one liter per centrifuge run.  Maybe his centrifuge was even smaller than in the paper.  Note the paper has a floor size shaker incubator for the liquid runs which weights 400 pounds.  The paper had much better equipment than Ivins had.  That means Ivins run times would be much longer and his chances to have dud runs or low yield runs much higher.

From the first run having only 10 percent spores according to Ed’s source, we can infer that they needed to grow.  That means the second run had to be grown between Sep 18 and Oct 6-9.

If those took 7 to 14 day runs plus centrifuging multiple times, etc. then the work likely started right after Sep 18.  So it wasn’t Ivins.

It seems on the first run, someone was in a hurry to dry it.  This is more consistent with someone who unexpectedly had to deal with a short time horizon.  If Ivins had a small centrifuge for smaller jobs than one liter than he couldn’t have really expected to dry 5 liters or more.  So he wouldn’t have tried at all.

Dont’ forget the subtilis that was different from Ivins lab.  It is likely a trace of subtilis was present at the start of the run and that this grew along with the anthracis.  That argues that a smaller amount of anthracis was used to start and that there had already been several days of growth, i.e. it wasn’t a run from Sep 14 to Sep 15/16 but a run somewhere else started more like Sep 11 or even a few days before.

==

http://www.druckerco.com/drucker/centrifuges/horizontal.asp

Top of line unit appears to be 300ml capacity.  The dtic paper did 30 minute centrifuge runs and did multiple ones.  So for 5 liters, which is the minimum size run he was doing, it would take 6.5 hours.  But he was only in the lab 2 hours 15 minutes each day the first weekend.  So there wasn’t enough time to centrifuge even once.

In a BSL3 lab, you don’t want giant centifuges.  When you have to clean it, and its when not if, you want as small a one as possible.  Also you can’t throw it out instead of cleaning it.  Everything has to be cleaned before being thrown out.  You want small equipment that is robust and won’t break, not giant centifuges that are going to have repair and break down problems and need a technician to repair who is an expert on that unit but unused to the BSL3.

==

The dog that didn’t bark in the night.  Ivins spent only 2 hours 15 minutes each on Fri Sep 14 to Sun Sep 16 by FBI’s own affidavits.

The material that was sent was not sufficiently centrifuged and washed to separate the spores from the rest.  This implies the person who sent it had run out of time.

But Ivins had loads of extra time.  If Ivins had a centrifuge on those days, then he could have spent extra hours centrifuging and washing.  If he didn’t have a centrifuge in his lab, its hard to see how he ever did it.

It appears one of the letters was more thoroughly prepared to cause inhalation anthrax.  So if Ivins was doing this and did that one better, why didn’t he do another one better too?  He had the time.  Lots of it.  But he didn’t.  This is the dog that didn’t bark in the night.

Ivins didn’t do the Florida run and the New York runs at the same time on this weekend.  But that was the FBI’s whole argument.  Moreover, Ivins had time that last weekend to do at least one letter better but didn’t.  So he wasn’t in there just doing the letters.

==

Lets not forget the subtilis contamination.  That indicates a test run was done with the equipment first.  That equipment was at least rinsed presumably.  Then the subtilis had a growth phase is likely.  So there was probably a growth phase of at least a couple days and possibly 7 days.

If we look at the paper I reference before some runs go 14 days.  They tested the runs at the end of each day and stopped when they reached a conversion of food to spores.

The people doing the run may have counted on 3 to 7 days and then they got a slow run and they went with what they had.  So they started say Sep 11 to mail Sep 17/18 and then got to Sep 16 or so and went with what they had.

One theory (Ross Getman or Kenneth Dillon?) is that there was a transfer in Maine by Atta to someone else that took place on Sep 11.  That would then explain their hoping to get a good run before Sep 17/18 but it not working out as shown by many runs in the dtic paper that don’t.

So one scenario is the subtilis prep run was done before Sep 11.  That ran in under 7 days.  Then that group got the anthracis from Atta who got it from someone who got it from a US site that had it.  Then the anthracis run was slow so they went with what they had.  Then they started their next run on Sep 18 and got it ready to mail Oct 6-9.

==Comment posted at following thread at Meryl Nass

http://anthraxvaccine.blogspot.com/2009/01/more-pictures-complete-exosporium.html

This is a good dialogue with good contributions by many.  Ed Lake is to be commended for his efforts and taking it in stride as have others.

The more steps, time taken, resources used, ideas exploited, money, etc. the more it indicates it was many who did it and not Ivins.  Its clear that the silicon fits that.

One test is to look for spores with silicon in the BSL3 or look for silicon on equipment that might have been taken out.  If he had leakage into BSL3 in processing then there should have been spores in BSL3 with silicon.  Or there should have been silicon left from the processing.  This could be under equipment, desks, etc.

Also if he took a lot of spores from the RMR1029 flask to speed up production, used silicon in processing and then put some spores back in RMR1029 to tank it back up, then there would be silicon back in RMR1029.  It appears there is none.  That means he likely didn’t add back to RMR1029.  That means his ability to use a large starting amount from it is less.

This means he needed more growing time to produce his runs.  This is very important for both sets, the Sep 17-18 and the Oct 6-9 mailings.  The former was on a tight schedule and did have .5 grams of bacillus anthracis spores according to some estimates.

The second run had at least 2 grams of spores.  So he had to grow this and he couldn’t speed up the growing by using more from the flask and then returning it.  That is very important.  Because it means he needed longer run times.

http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA426293

Carey, Laurie F. ; St. Amant, Diane C. ; Guelta, Mark A.

Is the paper with times for production.

For an external party, not Ivins, putting spores back in their source would not be an issue and we don’t have their flask.  For them it would make sense to use silicon to grow.  Ivins, however had reasons not to.  One is returning to the flask.  The other is it would leave a signature of spores in BSL3 that could be detected and finger him.  But it appears no such spores have been found in BSL3.  It seems almost impossible that Ivins worked quickly to do these large volume runs without leaving spores in the lab containing the same silicon.  Thus this is another proof that Ivins didn’t do it.

The subtilis contaminant is another proof he didn’t start with more from the RMR1029 and then put some back.  So we have to count on longer runs from smaller numbers of starting spores if Ivins did it.  Together with the dtic paper linked to above, that implies Ivins didn’t have the time is the reasonable interpretation of the evidence.

Also there should be some subtilis spores in the BSL3 if he prepared it there.  Some of those should have silicon too it would seem.  Those also were not found and that’s more evidence it didn’t happen there.

==

Given the large production volumes and the short time and ad-hoc equipment that was likely not up to the volumes done so requiring mutiple use, there should have been spores of anthracis and subtilis spiked with silicon all over BSL3.  None have been found.  That is pretty strong evidence the processing didn’t happen there.  Not with make shift methods of drying, processing, etc.  Not with feet of plates all over the place if he used plates.  Not with drying that didn’t have an adequate hood.  Etc. Etc.

==
Also the silicon was in both runs.  So it should have been
in the BSL3.  Since the silicon was in both runs, his ability
to start with a larger amount to speed up a run wasn’t possible
with either run.  So that method is subject to a sum inequality,
i.e. the amount taken out from his flask in sum was limited
by whatever bound we have on the decrease, at most the flask
size minus the amount in it being one estimate.  (There is
still remove and add water to consider?)

His better method was to start with more from the RMR1029 flask not
use silicon and then put back some into his flask.  Both times
that was his better method.  After the first failure, he should
have tried that the second time.

For an outside scenario, they could start the second run
on Sep 18 and get it done in time and do a precision job,
taking as long as needed for each step to get it right this
time.  The date Oct 6-9 when mailed was when it was ready.
Ivins could never feel such freedom.

The fact of a second run
with everything done right by itself is unlikely for Ivins
since he would never have the feeling that he could afford
to do each step of the procedure until that step was done
right and then pick up the next step.

In fact, each step would
have to end or be interrupted for the need of concealment.
His lab times are insufficient for each step to be done
perfectly.  He couldn’t produce a perfect run with a start stop
limitation over the last week, Friday to Friday.

==

If the spores he cleaned up in his work area outside BSL3 had silicon in them, shouldn’t there be some traces still?

Anthrax spores indels morphs FBI arithmetic

December 28, 2008

Vahid Majidi is the head of the FBI’s WMD unit.  He has put forward the theory of indel identification based on the work of himself and his team including outside scientists.  Jacques Ravel is one of the scientists who has worked on this.

However, there are assumptions and pitfalls in this claim that may result in the whole thing being junk science, i.e. they can’t identify RMR1029 the flask at Ft. Detrick of Bruce Ivins as the source to the exclusion of the other 1000 flasks tested or some subset larger than 8.

In 1981, Ames strain was isolated from a single cow.  That cow likely ingested anthrax spores from the soil or water, became sick and died.  Some of the anthrax was then sent on in the form of a blood sample or ear.  The lethal dose in humans for inhalation anthrax is often stated as 10,000 spores.  For a cow to ingest it, the dose didn’t show up in internet searches.  Cattle farmers and human volunteers for determining the exact lethal dose in cows and humans seem to be scarce.

The FBI claims to have asked 1000 labs to send samples.  From these they grew anthrax colonies and allegedly identified which colonies showed different appearance, morphs, and then from that identified indels, changes in the DNA molecule.   Whether that really is true is something the FBI needs to prove.

If they grew 1000 colonies from each lab, that is the same as testing 1000 spores for the indels.  Within a spore how many DNA molecules are being tested?  We can assume its one for now, but it is really more.  This introduces an additional complication, which we ignore for now.

The FBI claim is that the original Ames strain had one morph despite the cow having ingested more than one spore to get sick and die.  The cow might have ingested a microgram of anthrax spores.  1 gram has 1 trillion spores, so a microgram has 1 billion spores.  If the cow ingested 1 billion spores, and the FBI tested 1000 labs x 1000 colonies (= spores) each then the FBI tested 1 million spores total v. the 1 billion spores the cow ingested in that scenario.

The FBI claims that 8 morphs evolved from 1981 to 2001 from the Ames Strain and that it tested for 4 and that the original Ames spore sample had one single indel type in it.

Alternative Hypothesis: All 8 morphs the FBI has found of indels were present in the 1981 sample of spores that killed the cow and were eventually transmitted to the US research community.

Under the FBI hypothesis, 8 indels evolved in 20 years with a small amount of anthrax in labs.  But shouldn’t nature over millions of years have evolved a lot more?  If 8 indels are produced every 20 years in the labs with a few small runs, then shouldn’t nature have produced a staggering number of indels in wild anthrax?  This can be tested by testing wild Ames anthrax for how many indels there are, and whether they include the 8 the FBI has found in lab anthrax from the single Ames source.

Is it really true that no more wild anthrax was added to the lab stocks and thus added wild indels grown over the last million years on the entire planet earth?

One comparison to keep in mind is the 1 million spores tested by the FBI total, or perhaps 100 thousand to 10 million tested to what the cow ingested, which could have easily been 1 billion spores.

What anthrax colonies look like:

http://www3.niaid.nih.gov/NR/rdonlyres/DD661CCD-4E1E-4918-832B-DA66873C6037/0/ANDB3E_1.JPG

To test 1000 labs, they grew 1000 colonies each for a total of 1 million colonies?  Then they looked at them to identify the strange ones?  If one looks at above photo of anthrax this seems preposterous.  One person did the looking or many people?  At the source labs or at a central lab?  Someone at a central lab looked at 1 million colonies?  How long did that take?  Aren’t these differences in colony appearances subjective?  Don’t colonies grow randomly?  Isn’t that how the colony photos on the Internet look?

==

If they claim they do a million colonies per lab flask source, then how do they inspect them?  1 second each is 1 million seconds per lab flask.  3600 seconds in an hour, so about 300 hours per lab flask.

If they claim they can look at them all at once and see the morphs, then is that believable given the photo above?  If one has a million they aren’t all flat one colony layer thick.  So you can miss them behind them.

Once still gets statistics issues if one of the indels has a low probability frequency.

There is also the issue of how much variation there is in colony appearance from other factors.  Are they fooling themselves on the link from indel to morph appearance?  Isn’t there additional random variation?  Is the causal relation tested?  As a statistical relation?  Have they formulated classifications of the variation of visual appearance for the same indel?  Can they use statistics for that?  How do they do statistics if all identification is visual, just looking at a million colonies at a time?

How do they manage all this without errors creeping in?  How do the get it into numbers on computer hard drives to keep track of?  What data is on computer hard drives?  What isn’t?

Can they simulate the entire process from the cow in 1981 to present including the testing and the statistical variation in everything?

Rough Notes:

http://www.ph.ucla.edu/epi/bioter/theamesstrain.html

lethal dose of anthrax is 10000 spores?


A lethal infection is reported to result from inhalation of about 10,000–20,000 spores, though this dose varies amongst host species.[13]

lethal dose in stomach of cow is more than inhalation anthrax
in humans?

and it can eat more than the minimum lethal dose.

so it ate 1 million spores?

and those had more indel morphs than testing
10000 colonies from a lab.

1000 labs have lab assistants from anywhere in the world.

who don’t care.

http://www.ph.ucla.edu/epi/bioter/theamesstrain.html

cow ingested at least 10,000 but more likely 1 million
to 1 billion spores.

http://www.vetmed.ucdavis.edu/vetext/INF-BE_cca/INF-BE_cca01/INF-BE_cca0112.html
http://www.cattletoday.com/archive/2001/November/CT177.shtml

== More rough notes

equation author:J  author:Ravel

author:v-majidi

==

compartmentalization and lack of understanding what doing.

==

If they grow 1000 colonies from a lab flask, that is more work to identify the morph ones.  There must be random variation in the appearance.  Different people are doing this work.

From NYT

“Then an Army microbiologist from Fort Detrick made an unexpected discovery. Using an old-fashioned microbiological technique, he spread out some attack spores on a bed of nutrient and let each form its own colony. All the colonies looked identical except one, which, to his trained eye, seemed very slightly different. Different-looking colonies are called morphotypes or just “morphs.”

“Had that task been assigned to someone less experienced, these morphotypes might never have been seen or their significance never realized,” Dr. Fraser-Liggett said.”

So out of the 1000 labs tested, some had untrained eyes looking at the colonies grown.  If each lab grew over 1000 colonies that is a problem for untrained eyes.  Also compartmentalization made it harder to achieve consistency.  People would need to be trained what to look for.

So if they grow 1000’s of colonies per lab flask, then untrained eyes will glaze over to miss the perhaps single morph colony to be found.  If they grow under 100 colonies they miss growing the morph colonies because the article says they are under 1 percent each.

==Some posts at Meryl Nass on this

https://www.blogger.com/comment.g?blogID=6592607595936297457&postID=4547196115304895388&isPopup=true

http://anthraxvaccine.blogspot.com/2008/12/new-details-from-genomic-forensic.html

To avoid confusion, the FBI or someone has to give names to each morph and list in a table which ones were in which letters.

Its possible that there is also not a unified definition of what 4 variants means in the project. Some people think it means some sort of original plus 4 additional and others think that 4 means the 4 named morphs that are distinct from the original.

They should also have a photo of each colony and its name and explain why they think they look different from other morphs and what the variation of appearance is when the same morph is present.

Also, what are the percentages of each morph in the original flask and in the other 7 flasks (or is it 8?) that have it. Give the name of each lab that has the same batch as at Detrick.

Also what about other morphs they decided not to pursue? Science is supposed to falsify hypotheses not just convict dead men. How about a second team tries to break the first one’s results by finding other morphs not in the Ft. Detrick flask but in someone else’s?

December 26, 2008 2:02 PM

Old Atlantic Lighthouse said…
What about a single morph in one letter that is only in one lab flask? Or is in a lab flask different than RMR1029 and not in RMR1029? Those cases should be pursued next. Basically, any morph in any letter is back tested against all the flasks. Publish the entire table.

December 27, 2008 4:22 PM

Blogger Old Atlantic Lighthouse said…
The FBI’s thinking is: We know in advance the letter anthrax all came from a single flask, that of the lone nut. They decided that in advance, and their methodology builds that in. However, if one drops that assumption, there are other possibilities. Two or more sources sent independently. Two or more with mixing after powder is produced, possibly because not enough, and so on.

December 27, 2008 4:26 PM

Blogger Old Atlantic Lighthouse said…
“he spread out some attack spores on a bed of nutrient and let each form its own colony. All the colonies looked identical except one, which, to his trained eye, seemed very slightly different. Different-looking colonies are called morphotypes or just “morphs.””

You can’t tell until you grow them if they are different. So the anthrax in the letters can contain any number of morphs. Unless you grow all the anthrax in a letter you can’t know if you got all the morphs in the letter. So we have to have a stopping rule. One stopping rule is do all. But they probably kept some.

“Dr. Ravel was asked to decode seven more morph genomes,”

So 8 morphs total.

But there could be more.

The entire procedure had a stopping rule of convicting a lone wolf scientist. Stop growing and testing and comparing when you do that. But that is not trying to disprove your hypothesis.

December 27, 2008 4:39 PM

Blogger Old Atlantic Lighthouse said…
Maybe the 8 labs that found all 4 morphs were the 8 labs that grew 1000 or more colonies? The other labs grew fewer colonies? Monte Carlo was used at Los Alamos early on. Vahid Majidi is from Los Alamos.

We need a table of how many colonies grown by each lab and which morphs they found. We also need to know how many times they went back to grow more colonies from the Ivins flask until they got all 4 from that flask they were looking for, and whether they decided which of the 8 to look for after they concluded 4 were not in the Ivins flask.

December 27, 2008 7:25 PM

Blogger Old Atlantic Lighthouse said…
“Though 99 percent of its spores were identical with the Ames ancestor, some 1 percent or less were morphs. ”

” As the morphs became available, the F.B.I. started testing samples. At first, some had one or two of the morphs. None had three of the morphs.

By late 2005 to 2006 it became clear that just eight of the 1,070 samples collected included all four morphs.”

Because they started growing more colonies when they didn’t find all 4?

Let p[i][j] be the percentage of morph i in flask j. This is not a constant of nature. A flask can have a low percentage but still be there. If one grows 1000 colonies and p[i][j] is .01 in one flask and
.0001 in another, then you may not get it when testing that flask.

If a person was the bad guy he could just grow fewer colonies from his flask and he would not get all 4.

What about the Ivins sample that didn’t test out? Which morphs if any did it have, how many colonies grown, etc.

How long have these morphs existed? How often do they form on their own? The same morphs are formed easily all the time? They all come from a single Ames lab source from 1981? Or other wild Ames has been added to lab stocks which had the morphs from hundreds or thousands of years ago?

If all lab Ames is from a single 1981 source, then wild Ames should have lots of different morphs. That could be tested.

December 27, 2008 8:51 PM

==

.99^99 = .37

Suppose .5 taking into account error to see it.

1/2 for each of the 4.  so 1/16.

and prob is under .01

of all 4 under .01

then say

.0025

.9975

1/4 * 1/100 * .9975

so more like 1/10 to see one.

in that case, its 1 in 10,000

.9975^99 = 0.78050831

16/1000

.0016^.25 = 0.2

so if it was .2 to see one of them, then
to see 4 is .0016 and out of 1000 labs that is 16.

.008^.25 = 0.299069756

.3

so if the chance to see one of the colonies is .3 per lab flask
then we get odds of 8/1000 to see all 4.

so out of 1000 flasks we get 8.

==

Suppose taking into account all factors, the percentage in the flask, the colonies grown, error in seeing the few morphs, the chance is .3 per morph per lab flask to see a morph.  Then to see 4, one takes
.3^4 which is .0081.  So out of 1000 labs, one finds all 4 8 times.  In this case, all of the labs had the 4 morphs just only 8 detected them.

The total of all 4 morphs from the NYT article appears to be under 1 percent, so .25 percent each.  This might work out to the .3 chance to see a morph depending on the number of colonies grown and accuracy to identify the few morphs.

==

number of colonies grown is likely random.

and multiple spore source is also random.

and may impact colony appearance.  which one dominates if
different?  or its unique?

==

actually, need to consider

1000, 4 type factor as well?

it should average out?

compute average and standard deviation.

average is

np?

==

the indels have been there since 1981 and
are the same in Wild Ames?  they evolved thousands
of years ago.

think of how many would exist in the 1981 source
if 8 had evolved in 27 years.

if they evolved that fast in a few runs, then
evolution would have produced thousands of them in the
original Ames strain.

==

If 8 morphs had evolved from a single strain from 1981 and the 1981 had one only it doesn’t make sense.  If 8 evolve in 20 years to 2001, then the 1981 Ames should have had many many indels already each producing different colonies. I.e. evolution should have produced more than 1 indel in some sample taken in 1981.

It makes more sense that the 8 were all there in 1981 and that the chance to see each morph and record it correctly is .3 per morph per lab flask, so that 8 were seen randomly.  The letters were handled with more care and more colonies grown perhaps, so they identified those somewhat more accurately despite small fractions of the indels.

100bp = 1 percent = .01

1bp = .0001

= 1/10,000th

1ppm = .01 bp = .000001

the 4 morphs could be

.005,.003,.002,.001

ie 50bp,30bp,20bp,10bp

or

50bp, 10bp, 1bp, 1bp

or

5000ppm,1000ppm,100ppm,10ppm

if 1000 labs look at 1000 colonies that is 1 million colonies.

if a morph is in the frequency of 10ppm, then 10 labs see it
even though all have it.

If the original Ames sample was 10000 spores its a similar
set of statistics.    If the Ames was taken from one cow infected
by a single spore then it could have a single morph.  If
the Ames was taken from anthrax sourced from 1000 random spores, then its similar to 1000 colonies.  However, random wild spores should have more indels since there has been a long time to create them.  This can be tested against wild spores today.

There is also the question of the identification of these
colonies by sight.  Is that science or make believe?  Has the
method to identify colonies itself been tested?  And all
1000 labs used such a method?  Or its 1000 different people
looking at 1000 colonies each and making up their own rules
as to which colony is of a specified appearance?

==Following more repetitive rough notes including to some of the Meryl Nass posts

If you take anthrax powder and spread it out and grow it, isn’t it random which spores produce colonies?  So some morphs in the sample don’t grow to colonies and some do?  So it requires a statistical test?  Looks like the entire procedure needs to be tested by going forward and then backward under controlled conditions.  Also start with single flask or with multiple flasks with different morphs or overlapping morphs and see what happens.

Trillion spores anthrax only a few become colonies.

==

Each letter had about 1 trillion spores.  So they didn’t grow 1 trillion colonies, they grew say 10 to 100 or 1000.

Suppose out of the 8 morphs mentioned at one point, each is present in a sample (flask or letter) in the amount of one percent each.  If you grow 100 colonies from that flask or letter, then for those morphs you have a 1 in 100 chance to get it.

This applies to letters and to lab flasks.  They had 1000 labs they tested?  8 had the 4 morphs they were checking for?  If all 1000 labs had all 8 morphs but each lab grew 100 colonies per lab, then how many labs would have the 4 show up?

What if some labs grew 50 colonies?  What is some grew 10?  What if the Ivins flask, they grew 10,000 colonies and the other labs they grew 100 colonies?

There are quants from Lehman with nothing to do, why don’t they hire them to do Monte Carlo.

==
Maybe the 8 labs that found all 4 morphs were the 8 labs that grew 1000 or more colonies?  The other labs grew fewer colonies?  Monte Carlo was used at Los Alamos early on.  Vahid Majidi is from Los Alamos.

We need a table of how many colonies grown by each lab and which morphs they found.  We also need to know how many times they went back to grow more colonies from the Ivins flask until they got all 4 from that flask they were looking for, and whether they decided which of the 8 to look for after they concluded 4 were not in the Ivins flask.

==

compartmentalization helps you fix the science to the defendant.

Vahid has a good career ahead of him, on Wall Street.

Maybe the 8 labs that found all 4 morphs were the 8 labs that grew 1000 or more colonies?  The other labs grew fewer colonies?  Monte Carlo was used at Los Alamos early on.  Vahid Majidi is from Los Alamos.

We need a table of how many colonies grown by each lab and which morphs they found.  We also need to know how many times they went back to grow more colonies from the Ivins flask until they got all 4 from that flask they were looking for, and whether they decided which of the 8 to look for after they concluded 4 were not in the Ivins flask.

morph undel anthrax

how many spores produce a colony?  one?
or multiple?

multiple inheritence so to speak.

the appearance?  random walk or fractal or some
other stochastic complication?

unknown fractions of the undel dna in the
flasks and letters.

they don’t have the same frequency.

so number needed is variable.

==

“Though 99 percent of its spores were identical with the Ames ancestor, some 1 percent or less were morphs. ”

” As the morphs became available, the F.B.I. started testing samples. At first, some had one or two of the morphs. None had three of the morphs.

By late 2005 to 2006 it became clear that just eight of the 1,070 samples collected included all four morphs.”

Because they started growing more colonies when they didn’t find all 4?

Let p[i][j] be the percentage of morph i in flask j.  This is not a constant of nature.  A flask can have a low percentage but still be there.  If one grows 1000 colonies and p[i][j] is .01 in one flask and
.0001 in another, then you may not get it when testing that flask.

If a person was the bad guy he could just grow fewer colonies from his flask and he would not get all 4.

What about the Ivins sample that didn’t test out?  Which morphs if any did it have, how many colonies grown, etc.

How long have these morphs existed? How often do they form on their own?  The same morphs are formed easily all the time? They all come from a single Ames lab source from 1981?  Or other wild Ames has been added to lab stocks which had the morphs from hundreds or thousands of years ago?

If all lab Ames is from a single 1981 source, then wild Ames should have lots of different morphs.  That could be tested.

==

radiation causes the morphs?  or what?  stimulates them at
higher rate?  based on what is going on in the lab, altitude,
etc?

stochastic appearance of a colony?  multiple spore
origin of a colony?

other conditionds during growth, special appearance
depends on the indel and on one or more additional
factors?

==Possible Vahid Majidi responses:

Defense: We grow 1 million colonies per lab flask and use visual inspection.

Q: Did you take flasks that didn’t show colonies with the 4 morphs and see if they had the purported causal indels anyhow?  This could look at whether other factors are needed in combination to produce the purported different shapes (morphs)  or that the purported different shapes are just imaginary.   Stochastic processes are known to fool humans by producing patterns that are random.  Did you try to see if that was happening?  If so how?  How did you get data into computer files for statistical analysis if visual id was your method?

Pakistan 9/11 attack money then 9/18 anthrax attack diversion

December 14, 2008

Hypothesis: Pakistan.  Timeline: Week before 9/11/2001, the head of Pakistan’s ISI came to the US and met with US leaders to try to get sanctions lifted.  That failed. He gave the go-ahead for 9/11 to al Qaeda in the DC area.

Then he met with US intensively following few days.  US agreed to life sanctions and pay money to Pakistan.  He then gave the signal to the anthrax team to mail the anthrax on 9/18.  Reason? 9/11 was to turn on US concern for al Qaeda and that they needed Pakistan’s help to get to Afghanistan.

Once they got the money they sent out anthrax made from spores from a US lab to turn US attention to domestic terrorism.  Once they got the money they wanted to turn attention away from Pakistan.  It basically worked.

Before 9/11, there was talk the US might lift sanctions on India but not Pakistan.  Pakistan already considered the sanctions unjust.  The head of ISI was a fundamentalist and unjust in that context means its not just ok to kill but whatever it takes is required to do to remove the unjust situation.

http://www.iht.com/articles/2001/09/07/edmansoor_ed3_.php


Pakistan:Leaving U.S. Sanctions in Place Would Be Grave
By Mansoor Ijaz, R. James Woolsey and James A. Abrahamson
Published: FRIDAY, SEPTEMBER 7, 2001

NEW YORK: The Bush administration is preparing to lift punitive sanctions imposed on India for its nuclear program. But clearing this hurdle to bolster political, economic and military ties with India while maintaining even sterner sanctions on Pakistan would be seen throughout the Middle East as discrimination of the first order. Islamabad would be pushed in dangerous directions, particularly toward increased reliance on its nuclear and missile programs.

Further crippling a weak Pakistan, perhaps to the point of state failure, would invite its myriad problems to spill over into other countries. Islamic militancy, arms and drug trafficking and religiously motivated sectarian violence could have devastating consequences for India’s economic prospects and cause trouble in important Islamic oil-producing countries. Risks to U.S. forces and interests in the Middle East would be a virtual certainty.

Also

http://www.highbeam.com/doc/1P1-27095601.html

Search Pakistan “sanctions lifted” 2001, and other similar searches.  Toss in nuclear, IMF, etc.  Pakistan’s central banker on 9/11 later wrote a book and said Pakistan was unable to continue w/o lifting sanctions.  That meant Pakistan would have to give up its nuclear program but India wouldn’t.  That would be “unjust”.

http://www.historycommons.org/project.jsp?project=911_project

has info on Pakistan and 9/11.  Can look up meetings ISI and US after 9/11 before 9/18.  Also has an anthrax timeline.

http://www.historycommons.org/project.jsp?project=2001anthraxattacks

By Sep 23 2001 Sanctions lifted in the news.

http://news.bbc.co.uk/1/hi/world/americas/1558860.stm

Pakistan knew that before 9/18/01.  There were also anthrax incidents in Pakistan in 2001 after 9/11.  Pakistan ISI was capable of getting anthrax from a US source and then producing it. Only Pakistan and the others involved in 9/11 could plan it all out in advance and be ready to mail on 9/18 from NJ and from Florida the fake letters to same people, e.g. NBC.

Only Pakistan had a motive to divert attention from 9/11 on 9/18 because then they had the money and sanctions lifted deal.  Means, motive, opportunity, timing.  It all fits Pakistan.

Pakistan also has the MO before 9/11 and up to now.  In 1999, Musharraf invaded Kargil and said Pakistani troops were terrorists.  India won and wanted to return the bodies of Pakistan soldiers, but Pakistan still said they were terrorists and wouldn’t take the bodies of their own soldiers.

Above comment posted here:

http://anthraxvaccine.blogspot.com/2008/12/confirmation-letter-spores-grown-in.html

Problems with FBI theory from further results released:

http://www.nae.edu/NAE/pubundcom.nsf/webviews/Engineering+Innovation+Podcast+and+Radio+Series?OpenDocument&count=3

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