A published yield of B. anthracis Ames spores grown on solid medium is 8 × 109 spores per Petri 150-mm plate (Carrera et al., 2007). Production of the required number of spores on plates, with the conservative assumption of no spore losses during purification, would therefore require 463 to 1,250 plates. Expert testimony to this committee indicated that 15 liters of liquid culture in a fermentor could yield 2 × 1013B. anthracis spores (Heine, 2010). Cultivation in shake flasks or losses during spore purification could certainly reduce this yield severalfold. RMR-1029, was a well-characterized large-scale spore preparation housed at the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID). The initial 1-liter purified spore preparation in RMR-1029 was derived from approximately 160 liters of culture and contained an estimated 3 × 1013 spores. Thus, cultivation in the range of 2.8 to 53 liters of liquid medium would have been required to produce the spores required for the letters (see Table 4-2).
Spore purification is typically accomplished by repeated centrifugation, disposal of the supernatant-containing cellular debris, and resuspension of the spore pellet in fresh liquid. High spore purity is generally achieved by centrifugation of the spores through a gradient of a high-density compound. The most commonly used high-density compound, meglumine diatrizoate, was not detected in the letter material (see Section 4.6 below), suggesting that this procedure was not used or that the spores were extensively washed after such a procedure. Purification by any method would involve some liquid washing steps and would require a relatively large-capacity centrifuge. Such instruments are commonly found in microbiology laboratories. The spores in the Leahy and Daschle letters were accompanied by less contaminating debris, and thus were of higher purity and concentration, than those in the New York Post letter (FBI Documents, B1M2D2, B1M2D6).
There are several methods for drying spore suspensions to produce powders like those found in the letters: these include chemical desiccation, air drying, and freeze drying (lyophilization), any of which could require several hours to several days. Drying of surrogate spore preparations using various
|Total spores needed for all letters||Spores prepared on plates at 8 × 109 spores/plate||Spores prepared in liquid|
|Low estimate = 3.7 × 1012||463 plates||2.8 liters in a fermentor, based on Heine’s (2010) estimate of 2 × 1013 spores from 15 liters|
|High estimate = 1.0 × 1013||1250 plates||53 liters in a fermentor, based on RMR-1029 having 3 × 1013 spores from 160 liters|