Variables affecting two electron transport system assays

Abstract

Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4° C. Extraction efficiencies of formazan from sediment varied with alcohol type; methanol was unsatisfactory. Phosphate buffer (0.06 M) produced higher activity than did either U.S. Environmental Protection Agency reconstituted hard water or Tris buffer sediment diluents. Intracellular formazan crystals were dissolved within minutes when in contact with immersion oil. Greater crystal production (respiration) detected by a tetrazolium salt assay occurred at increased substrate concentrations. Test diluents containing macrophyte exudates produced greater activity than did phosphate buffer, U.S. Environmental Protection Agency water, or ultrapure water diluents. Both assays showed decreases in sediment or bacterial activity through time.