Making anthrax bacteria into a biological weapon requires milling the spores into particles small enough to ensure that they remain suspended in the air for long periods of time.
1) If they are grown on a culture medium, anthrax spores need to be dried. Because they are so durable, they can be freeze dried, heat dried or spray dried
2) Once the spores are dry, they are ground down to the smallest possible particle size, anywhere from one micron (one spore) to 20 microns. The process adds electrostatic charges to the particles, which makes them clump together
3) Chemicals such as bentonite or silica are added to remove the electrostatic charge and allow the tiny particles to float in the air, thus neutralising the spores
In order to cause disease, at least 8,000 to 10,000 spores need to lodge deep in the lungs, in the tiniest air sacs known as alveoli. The warm, moist environment, and possibly the concentration of carbon dioxide in the lungs, stimulates the bacterium to emerge from its protective spore. As each bacterium reproduces, it releases toxins, which eventually spread throughout the body and destroy tissue and organs.
Although the biosynthesis of saxitoxin seems complex, organisms from two different kingdoms, indeed two different domains, species of marine dinoflagellates and freshwater cyanobacteria, are capable of producing these toxins. While the prevailing theory of production in dinoflagellates was through symbiotic mutualism with cyanobacteria, evidence has emerged suggesting that dinoflagellates, themselves, also possess the genes required for saxitoxin synthesis.
Saxitoxin biosynthesis is the first non-terpene alkaloid pathway described for bacteria, though the exact mechanism of saxitoxin biosynthesis is still essentially a theoretical model. The precise mechanism of how substrates bind to enzymes is still unknown, and genes involved in the biosynthesis of saxitoxin are either putative or have only recently been identified.
Two biosyntheses have been proposed in the past. Earlier versions differ from a more recent proposal by Kellmann, et al. based on both biosynthetic considerations as well as genetic evidence not available at the time of the first proposal. The more recent model describes a STX gene cluster (sxt) used to obtain a more favorable reaction. The most recent reaction sequence of Sxt in cyanobacteria is as follows. Refer to the diagram for a detailed biosynthesis and intermediate structures.
1) Load the acyl carrier protein (ACP) with acetate from acetyl-CoA, yielding intermediate 1.
2) Follow up with SxtA-catalyzed methylation of acetyl-ACP, which is then converted to propionyl-ACP, yielding intermediate 2.
3) Perform another SxtA a Claisen condensation reaction between propionyl-ACP and arginine, producing intermediate 4 and intermediate 3.
4) Transfer SxtG to an amidino group from an arginine to the α-amino group of intermediate 4 producing intermediate 5.
5) Intermediate 5 then undergoes retroaldol-like condensation by SxtBC, producing intermediate 6.
6) SxtD adds a double bond between C-1 and C-5 of intermediate 6, which gives rise to the 1,2-H shift between C-5 and C-6 in intermediate 7.
7) SxtS performs an epoxidation of the double bond yielding intermediate 8, and then an opening of the epoxide to an aldehyde, forming intermediate 9.
8) SxtU reduces the terminal aldehyde group of the STX intermediate 9, thus forming intermediate 10.
9) SxtIJK catalyzes the transfer of a carbamoyl group to the free hydroxyl group on intermediate 10, forming intermediate 11.
10) SxtH and SxtT, in conjunction with SxtV and the SxtW gene cluster, perform a similar function which is the consecutive hydroxylation of C-12, thus producing saxitoxin and terminating the STX biosynthetic pathway.
Ricin is insoluble in normal water solutions, but combines well with slightly acidic water or lemon juice. It may be dissolved in water at a pH of 3.8 to 4,0 for injection or finely powdered for ingestion or inhalation. Its only drawback is that it is heat liable, meaning it will be inactivated by heating to over 100° C when in solid form or 60° C to 70° C in solution. The heat of grinding will also destroy the toxin, but if you gently crush it on a glass surface, using the back of a spoon or similar instrument, no deactivation should occur. Even with this limitation it is as close to a perfect poison as is available. It is odorless, taseless, undetectable. untreatable, and fatal in minute dose. Gram for gram ricin is deadlier than most nerve gases. Anything with this much power should be handled with the utmost caution and respect. This tutorial will show 2 methods of creating ricin:
How to make it:
Using acetone, dilute sulfuric acid, sodium hydroxide (lye), sodium carbonate (washing soda), sodium sulfate, and carbon tetrachloride. Uses more chemicals and equipment but produces an article of much greater purity. All chemicicals used are cheaply and readily available. Geared to the production of larger quantities than field grade.
1) Pour the whole beans into the blender. Add just enough acetone to cover them and grind for one minute. Check to make sure that all beans are ground up and that the temperature has not risen too much.
2) Add four times as much acetone as bean pulp to the blender and liquefy for several minutes.
3) Pour off the acetone and replace with an equal amount. Liquefy again for several minutes. Discard the used acetone.
4) Filter the slurry and allow the filtrate to dry thoroughly.
5) Add filtrate to blender with four times as much distilled water at a pH of 3.8 and a temperature of 25°C. Liquefy for several minutes. Note: the preferred pH range is 3.5 to 22.214.171.124 is optimal. 5% sulfuric acid is preferred for pH adjustment, although dilute hydrochloric acid can be used.
6) Filter slurry and discard filtrate.
7) Raise pH of this solution to pH 7 to 8, using 5% sodium hydroxide or 12% sodium carbonate.
8) Treat the solution with a 16.7% solution of sodium sulfate (2 pounds of salt to 10 pounds of water) to precipitate the toxin. Add a little at a time and cease additions when no more toxin is precipitated. Allow up to 5 minutes between additions.
9) Filter solution and discard the liquid. Wash filtrate with some of the sodium sulfate solution. This will remove an additional 15% non-toxic nitrogen.
10) The filtrate is dried and slurried with carbon tetrachloride to separate the ricin by flotation. Use caution handling carbon tet as it is a suspected carcinogen and has toxic fumes. The ricin is skimmed off the top. Dry and grind carefully.
11) Ricin is added to three times its weight of distilled water and brought to a pH of 3.8, using 5% sulfuric acid.
12) Filter the slurry and neutralize the pH by adding 12% sodium carbonate solution, a little at a time, until a pH of 7 to 8 is reached.
13) A second precipitation is brought about by adding the sodium sulfate solution. A precipitation time of 45 minutes is required.
14)The solution is filtered and the ricin is washed on the filter with sodium sulfate solution to remove additional non-toxic nitrogen.
15) The filtrate is dried, ground carefully, and slurried with five times its weight of carbon tetrachloride to separate the sodium sulfate by flotation. Skim the ricin off the surface. The nitrogen content is then reduced from 40 to 50%, to 15 to 18%. 16) Dry and carefully grind into a fine powder.
Store in a well-sealed container and protect from heat. Note: The grinding steps are the Achilles heel of this operation. The heatgenerated by grinding can easily deactivate the toxin. An air grinder was developed that eliminated this problem and may be available commercially. For best results the ricin should be as fine as possible.