Improved in vitro detection of hemolysin BL from Bacillus cereus

The production of a discontinuous hemolysis pattern that is characteristic of hemolysin BL, an enterotoxic and dermonecrotic hemolysin previously described, was investigated with 114 Bacillus cereus, two B. thuringiensis and nine B. mycoides strains. Discontinuous patterns were monitored by a blood agar gel diffusion assay of 5 h and overnight culture supernatants, and by direct examination after colony growth on blood agar. For gel diffusion, the condition for optimal discontinuous pattern development included the use of sheep blood agar containing 1–2 mM EDTA, the addition of calf serum (8% v/v) to supernatants before loading the gels, and incubating at 27°C; under these conditions the patterns generally appeared within 4 h of incubation. The final percent of strains exhibiting a pattern was 74–78%. For the colony system, two types of blood agar were used: nutrient agar (NA) and brain heart infusion agar with glucose (BHIG) containing 8% calf serum and 0.1–1 mM EDTA. Both NA and BHIG, containing 0.1 mM EDTA and incubated at 22°C, gave the highest percent of strains exhibiting a pattern. The patterns generally appeared between 12 and 28 h. Used in combination, NA and BHIG gave 73–74% of positive B. cereus/thuringiensis. Previous results obtained with 68 strains (Beecher and Wong, 1994a,b) were confirmed; the gel diffusion system was improved regarding homogeneity of time range of development, clarity, stability and final yield of patterns; the conditions for the colony growth system were chosen to make monitoring compatible with a laboratory working schedule. In both cases central hemolytic zones, that can hamper the observation of the patterns, were decreased. It did not seem that B. cereus, B. thuringiensis and B. mycoides can be differentiated on the basis of production of hemolysin BL.