An Improved Method for the Production of Bacillus subtilis var. niger Spores for Use as a Simulant for Biological Warfare Agents-Quality Analysis

Standards for proliferation of Biological Warfare BW agent simulants for use in development of detection and identification equipment are essential. Lack of standardized protocols for growth, processing, and product characterization will likely lead to variances in growth parameters and could induce changes in simulant characteristics that may affect instruments being developed. We have evaluated thirteen media purported to grow spores against several criteria, including growth time, ease of processing, reproducibility, and component definition. The goal is to have a chemically defined medium that will produce whole bacillus spores in the least amount of time. Three media were selected for further testing. Spores produced from each medium were tested for viability counts, purity, particle size, identity, and appearance under electron microscope. The spores exhibited similar viability counts per dry weight, do not contain extraneous organisms, and are virtually free of debris. Particle size is comparable when grown on each medium and spores are easily aerosolized and measured by an Aerodynamic Particle Sizer APS. Identity testing using infrared spectroscopy and PCR indicate positive identity with no major interference factors. Data indicates suitability for use in BW agent detector development.