IDENTIFICATION OF HEMOLYSIN BL-PRODUCING BACILLUS-CEREUS ISOLATES BY A DISCONTINUOUS HEMOLYTIC PATTERN IN BLOOD AGAR

Bacillus cel eus causes distinct exotoxin-mediated diarrheal and emeti c food poisoning syndromes and a variety of nongastrointestinal infect ions. Evidence is accumulating that hemolysin BL is a major B. cereus virulence factor. We describe two methods for detection of hemolysin B L in crude samples and on primary culture media. In the first method, the highly unusual discontinuous hemolysis pattern that is characteris tic of pure hemolysin BL was produced in sheep and calf blood agar aro und wells filled with crude culture supernatant from hemolysin BE-prod ucing strains. In the second method, the pattern was formed surroundin g colonies of hemolysin BE-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood . Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B. cereus isolates and in 46 of 136 (34%) presumptive B. cereus isolates from soil. All nine isolates tested tha t were associated with diarrhea or nongastrointestinal illness were po sitive for hemolysin BL. The methods presented here are specific, simp le, inexpensive, and applicable to the screening of large numbers of s amples or isolates.