WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line Puriya Ngamwongsatit a , Padmapriya P. Banada b , Watanalai Panbangred a , Arun K. Bhunia b, ⁎ a Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand b Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, IN 47907, USA ABSTRACT ARTICLE INFO Article history: Received 10 August 2007 Received in revised form 4 March 2008 Accepted 5 March 2008 Available online 13 March 2008 Keywords: Bacillus cereus MTT WST-1 Cytotoxicity CHO 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 (R 2 = 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. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The genus Bacillus contains a diverse group of pathogenic and harmless environmental species. Of which bacteria belonging to “Bacillus cereus group” are closely related, and are comprised of B. anthracis, B. thuringiensis, B. cereus and others (La Duc et al., 2004; Barker et al., 2005; Hoffmaster et al., 2006; Castanha et al., 2007; Leoff et al., 2008). B. anthracis is the causative agent of anthrax while B. thuringiensis is an insect pathogen and some of which also produce enterotoxins. B. cereus is an opportunistic pathogen and it is re- sponsible for systemic infection leading to pneumonia, endopthalmi- tis and bacteremia (Kotiranta et al., 2000), and emetic and diarrheal food poisoning (Schoeni and Wong, 2005). B. cereus is often present in a variety of foods such as rice, pasta, spices, milk and dairy products, vegetables, meat, cakes and other desserts (Larsen and Jørgensen, 1997). B. cereus produces several toxins including emetic (Agata et al., 1995) and at least four other enterotoxins: hemolysin BL (HBL; Beecher and Wong, 1997), non-hemolytic enterotoxin (NHE; Lindbäck et al., 2004) and two single proteins, cytotoxin K (CytK; Lund et al., 2000) and enterotoxin FM (EntFM; Asano et al., 1997). These toxins adversely affect the metabolic status or induce membrane damage of cultured mammalian cells such as CHO, McCoy, HEp-2, Vero and Ped- 2E9 cells, which can be measured using different endpoint assays for cytotoxicity in vitro (Mikami et al., 1994; Beattie and Williams, 1999; Finlay et al., 1999; Lindsay et al., 2000; Rowan et al., 2001; Pedersen et al., 2002; Gray et al., 2005; Katayama et al., 2005). Such assays include the microscopic examination of cytopathic effects (Jackson, 1993; Fletcher and Logan, 1999), colorimetric detection of alkaline phosphatase release (Gray et al., 2005), and MTT (3-[4,5-dimethylthia- zol-2-yl]-2,5-diphenyl tetrazolium bromide)-based metabolic activity staining (Dietrich et al., 1997; Finlay et al., 1999). The MTT assay (Mosmann, 1983) was developed to detect the emetic toxin in B. cereus, using HEp-2 cell line, where the toxin in- duces vacuole formation in the mitochondria (Finlay et al., 1999). Later, this assay was used by others for detection of toxigenic Bacillus species (Pedersen et al., 2002; Rowan et al., 2003; Taylor et al., 2005). In recent years, MTT-based cytotoxicity assay using CHO-K1 cell line was used to detect both the diarrheagenic and emetic toxins (Pedersen et al., 2002; Toh et al., 2004; Gray et al., 2005). This assay is non radioactive and can be performed entirely in a microtiter plate and is now widely used for quantification of cell proliferation and cytotoxicity. Though the MTT assay is sensitive, it requires 52 h to 72 h to complete, which is undesirable in the food industry where pathogen detection must be performed quickly. Journal of Microbiological Methods 73 (2008) 211-215 ⁎ Corresponding author. Molecular Food Microbiology Laboratory, Department of Food Science, 745 Agriculture Mall Drive, Purdue University, West Lafayette, IN 47907- 2009, USA. Tel.: +1 765 494 5443. E-mail address: bhunia@purdue.edu (A.K. Bhunia). 0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2008.03.002 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth