A bacterial toxicity assay performed with microplates, microluminometry and Microtox reagent.

We have developed a procedure for undertaking a Microtox-based test by coupling microplate and microluminometric technologies. Sample dilutions are prepared in a 96-well polystyrene microplate kept at 15 degrees C, while the Microtox reagent and diluent are placed in an opaque, microluminometry-compatible 96-well microplate also kept at 15 degrees C. Exposure begins when sample aliquots are brought into contact with bacterial reagents in the opaque microplate. After specific exposure times (5, 15, 30 and 60 min), bacterial luminescence is rapidly measured by placing the opaque microplate in a microluminometer. Reproducibility of the procedure, as well as general agreement of EC50 end-point values with published reports, is demonstrated herein after toxicity trials with six metals (Ni2+, Cd2+, Zn2+, Pb2+, Cu2+ and Cr6+). Results suggest that a 60-min exposure time may have value in getting more "sensitivity mileage" out of this Microtox-based assay. This microplate procedure possesses attractive features that augment sample throughput and information output. Further refinement and validation studies are ongoing in our laboratories.