Activity of a “thermostable exotoxin” of Bacillus thuringiensis subsp. morrisoni in the Salmonella/microsomal assay for bacterial mutagenicity

The purpose of this study was to employ the Salmonella/microsomal assay (Ames test) to investigate the mutagenic potential of a thermostable exotoxin of Bacillus thuringiensis subsp. morrisoni. Bacteria are ideal for the detection of infrequently occurring point mutations because the large number of organisms (200 to 400 million bacteria per plate) exposed to the mutagen at any one time increases the possibility of observing a random mutational event. The exotoxin used in this study was produced using the shaker flask fermentation procedure with mineral casein broth. A Petri dish method of bioassay using fresh bovine feces was used to determine the efficacy of the exotoxin against horn flies. The LD 50 was found to be 5.35 μl/g of feces. Five bacterial tester strains were identified and characterized for the genetic markers described by Ames et al. (B. N. Ames et al., 1975, Mutat. Res., 31, 347–364). Appropriate doses of the B. thuringiensis supernatant, solvent or positive control were added to agar plates. The supernatant was tested at five dose levels against all five strains of bacteria. Controls of bacteria only were included for spontaneous reversions. All treatments were performed in triplicate. The numbers of revertant colonies from each set of triplicate plates were averaged and the standard deviation calculated and compared to that found with the solvent control. The negative controls, positive controls, and sterility controls all fulfilled requirements for determination of a valid test. No detectable mutagenic activity was found for the thermostable exotoxin of B. thuringiensis morrisoni.