WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line.
Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2-3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P=0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44-52 h. A positive correlation (R2=0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.
Ngamwongsatit
Puriya
P
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Banada
Padmapriya P
PP
Panbangred
Watanalai
W
Bhunia
Arun K
AK
D000818:Animals; D001407:Bacillus / Q000472:pathogenicity*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000633:toxicity*; D001431:Bacteriological Techniques / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005516:Food Microbiology; D013778:Tetrazolium Salts / Q000378:metabolism*; D013844:Thiazoles / Q000378:metabolism
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D001427:Bacterial Toxins; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue