The tetrazolium reduction method for assessing the viability of individual bacterial cells in aquatic environments: improvements, performance and applications

Abstract

The electron transport system of respiring organisms reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Active bacterial cells may be recognized under the microscope by epifluorescence and by the simultaneous presence, seen under bright light field of optically dense intracellular deposits of INT-formazan. An improved procedure that leads to a sharp definition of cells and formazan deposits is presented here. Cells are concentrated on cellulose membrane filters of 0.1 µm porosity which are rendered further transparent prior to immersion of the cells in a layer of 4′, 6-diaminidino-2-phenylindole (DAPI) s′ fluorochrome. This process leads to two significant improvements: (1) the fluorochrome is not trapped inside the membrane, which decreases the background fluorescence and leads to a better detection of the small cells; (2) the cells are immersed in an aqueous solution, which prevents rapid dissolution of the formazan crystals which would be expected if they were in contact with oily clearing agents. Tests on formazan labelling and on storage of INT-processed samples suggest other precautions for reliable use. Improved in this way, the method is simple, rapid and has numerous applications in environmental studies, ecophysiology and ecotoxicology. Some examples are given, with 2 to 98% of INT reducing cells observed, depending on different environmental conditions.