Letters to the Editor
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@batman
- you can still vent to the outside safely with a HEPA filter (or 2 or 3 in series if you're paranoid)
- the double door doesn't have to be an air lock. The doors have to be on springs, auto closing, and the lab has to be under negative pressure. That means all air goes in, none out, except through a filtered system. This can be accomplished by sealing all the seams very well and putting a good vent system, such as the ones used to remove cigarette smoke from restaurants, on the roof.
- anthrax spores do not require as much purification as membrane proteins. Consider that membrane proteins must be over expressed, and have a tendency to form inclusions which cannot be purified. As you know, they also have to be isolated from the rest of the cell contents. Not so with anthrax. This is a viable pathogen that forms quantities of spores on its own (i.e. without genetic engineering) under environmental stress. Basically, anthrax spores are already pre-packaged, you just have to stress the organism to make it produce spores. You don't need any sort of column, much less an antibody.
- if you handle everything remotely toxic inside a safety cabinet (its awkward, but doable) you minimize risk to an acceptable level.
- I am constantly surprised people's ability to ignore serious risks. One man I worked with mouth pipetted everything, including acrylamide. The lab does not have to be up to regulation standards to be productive and under the radar.
wikipedia on anthrax: [excerpted]
The bacterium normally rests in spore form in the soil, and can survive for decades in this state. Anthrax in wild livestock occurs in the United States. Many workers are routinely exposed to significant levels of anthrax spores but most are not sufficiently exposed to develop symptoms. A lethal dose of anthrax is reported to result from inhalation of 10,000 - 20,000 spores. Cultivating anthrax spores can take minimal equipment and a first-year collegiate microbiological education.
wikipedia: bacillus anthracis
The bacterium can be cultivated in ordinary nutrient medium under aerobic or anaerobic conditions. B. anthracis spores are known to survive along livestock trails in the United States causing frequent outbreaks in states from Texas to South Dakota (CDC, 2001).
Furthermore, from a masters thesis by Darlene Sabio; Surface Characteristics of Bacillus Spores; College of Humanities and Sciences; Virginia Commonwealth University
downloaded as pdf from: http://etd.vcu.edu/theses/available/etd-05052004-135202/
Spores from laboratory stocks and new environmental isolates were compared using light and electron microscopy, total luminescent spectroscopy (TLS) and relative surface hydrophobicity and hexadecane partitioning.
Bacillus spores were germinated in BHI. [Brain Heart Infusion broth] Cultures were spread on sporulation agar plates and allowed to grow for one week at 37°C under aerobic conditions. They were harvested by scraping and suspended in sterile DI water. The suspensions were subjected to lysozymal degradation at 37°C for 2 to 12 hours, and then the suspensions were subjected to differential centrifugation three times at 9,000g for 15 minutes, and suspended in sterile DI water.
A negative stain was performed to assess the purity of the spores. If vegetative cells were still present, the lysozyme and centrifugation processes were repeated until the spore suspensions were free of vegetative cells (Riesenman, et al. 2000).
[Riesenman, P., Nicholson, W. 2000. role of the Spore coat Layers in Bacillus subtilis spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation. Applied and Environmental Microbiology 66(2): 620-626.] This reference also gives a similar description of germinating and growing the bacteria and of isolating the spores.
Neither source describes the quantity of spores derived from this process, but a number of things suggest that it was not minor.
- multiple species were considered in each study, suggesting that no extravagant effort was required for each species.
- there is no indication of the number of agar plates inoculated, suggesting that it is not extravagant.
- amounts appear to have been sufficient for several subsequent tests, although the tests carried out did not require large amounts of material.
At a minimum, this suggests that multiple mg of spores were isolated for each of 13 species as a fraction of the work involved in a 1-2 year masters' thesis.
Not quite a team of people in a campus-sized facility. Oh, and the thesis has some nice pictures of the various spores in the results section.
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@batman
I forgot about the centrifuges.
The masters thesis specifies 9000g for 15 min.
You can get that second hand (with the high speed rotor) in a floor model for about $3000 at http://www.labx.com/v2/adsearch/detail3.cfm?adnumb=319709
Actually, that is capable of far more than 9000g, and it would require a large biosafety closet. Maybe better to hold out for a benchtop model. Looks like the new sigma 3-18K with a 12150 rotor will do it, although it would cost a bit more. In both cases, the capacity is at least 1 liter, so it wouldn't require too many batches.
Also, you could get away with a slower machine if you're willing to run it for longer. Probably not a toss-away from an undergrad lab, but there are plenty of hospitals, schools and businesses replacing old equipment. A 1 liter capacity model that will go to 6000g or more isn't terribly rare, or specialized.
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The Media Today
For about three years beginning in l950 I worked for the news director (Hollis Seavey) at the MBS radio station in Washington, D.C. There were three or four correspondents on the staff, including Joe McCaffrey who covered the Hill. These were hard-working reporters with integrity. They never socialized with the powers that be in Washington. They lived in middle-class neighborhoods in Arlington and Falls Church, and they were truly the "working press." Today reporters are celebrities along with the people they cover, and are pictured together with well-known public figures at parties, galas etc. And we wonder why the press does not speak out as Americans expect them to do. I wonder if it is possible for the mainstream media to return to doing what we hv always expected- seeking the truth and being watchdogs for our freedoms. Perhaps the Web is our answer. Sincerely