A novel colorimetric yeast bioassay for detecting trichothecene mycotoxins.
A novel colorimetric microbial bioassay for toxicity has been developed; it shows particular sensitivity to trichothecene mycotoxins. The assay uses inhibition of expression of beta-galactosidase activity within the yeast Kluyveromyces marxianus as a sensitive toxicity indicator, cultures remaining yellow, rather than turning deep green-blue, in the presence of X-gal, a chromogenic substrate. The assay is conducted in standard microtitre plates, permitting small volumes (160 microl) and many replicates, and can be scored either automatically by a plate-reader, or by eye. Factors likely to affect the efficacy of the bioassay, including carbon source, solvents, inoculum cell density, and the use of membrane-modulating agents (MMAs), were assessed. Polymyxin B nonapeptide was the most effective toxicity-enhancing MMA tested, enabling the trichothecene mycotoxin, verrucarin A, to be detected at a concentration of about 1 ng/ml. The assay's reproducibility was examined using polymyxin B sulfate, a cheaper MMA, and another trichothecene mycotoxin, T2 toxin: reproducibility and sensitivity were better for the beta-galactosidase X-gal endpoint than for an alternative chromogenic toxicity indicator, the respiratory substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).
Engler
K H
KH
University of Greenwich, Woolwich, London, UK.
Coker
R
R
Evans
I H
IH
D001681:Biological Assay / Q000379:methods*; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology; D003124:Colorimetry; D007716:Kluyveromyces / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D011112:Polymyxin B / Q000031:analogs & derivatives / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013605:T-2 Toxin / Q000032:analysis / Q000633:toxicity; D014255:Trichothecenes / Q000032:analysis* / Q000633:toxicity; D001616:beta-Galactosidase / Q000378:metabolism*
D002593:Cetrimonium Compounds; D014255:Trichothecenes; C038934:polymyxin B nonapeptide; D001616:beta-Galactosidase; D013605:T-2 Toxin; D011112:Polymyxin B; C005348:muconomycin A; D000077286:Cetrimonium