A tissue culture assay for direct detection of sodium channel blocking toxins in bacterial culture supernates

A quantitative assay for sodium channel blocking toxins such as tetrodotoxin and saxitoxin has been developed for use with a microtitre plate reader. Mouse neuroblastoma cells, which die rapidly in the presence of ouabain and veratridine, were protected by tetrodotoxin; surviving cells were detected by their uptake of the vital dye Neutral red which was quantified with a microtitre plate reader at 540 nm. A sigmoidal dose response curve was obtained and tetrodotoxin concentrations were readily measured over the range 10 nM to 500 nM (3.2-160 ng/ml). With this method, sodium channel blocking toxins were detected directly, without processing or concentration, in culture supernates of several marine bacteria, including Shewanella alga, Alteromonas tetraodonis, Listonella (Vibrio) pelagia, V. alginolyticus, V. anguillarum and V. tubiashi. Culture supernates of Shewanella alga contained up to 510 ng/ml of sodium channel blocking toxin (using tetrodotoxin as a standard).