Toxic Effects on Bacterial Metabolism of the Redox Dye 5-cyano-2,3-ditolyl Tetrazolium Chloride

The monotetrazolium redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been used as a vital stain of actively respiring bacteria for several years. In this study, inhibitory effects on bacterial metabolism of this redox dye have been examined in a brackish water environment (Kiel Fjord, Germany) and a freshwater environment (Elbe River, Germany). As the results from time series experiments (1 to 10 h) show, bacterial growth and respiration of the investigated natural communities were clearly reduced by CTC supply. Compared with untreated controls (100%), CTC-treated samples showed distinctly lower heterotrophic bacterial plate counts (0 to 24 and 11.8 to 23.7%, respectively), bacterial production (0.9 to 14.1 and 1.1 to 9.6%, respectively), bacterial respiration (4.1 to 9.4 and 6.8 to 43.8% for several concentrations of (sup14)C-labeled glucose), and [(sup14)C]glucose incorporation (0.2 to 4.2%). Additionally, toxicity of CTC was demonstrated by luminescence in a Microtox bioassay. CTC concentrations of 0.1 and 5.0 (mu)M required only 15 min for decreases of approximately 50 and 100%, respectively. The suppression of CTC on several bacterial metabolic processes suggests that determination by the CTC technique underestimates the actual number of active cells distinctly. This conclusion is confirmed by the comparison of generation times calculated on the basis of thymidine uptake data and active bacterial counts determined by the CTC assay and microautoradiography. While unrealistic short generation times (0.5 to 5 h) resulted from the CTC assay, the generation times calculated according to microautoradiography ranged within values (7 to 21 h) reported elsewhere for comparable aquatic environments. The inhibitory effect of CTC demonstrated in our experiments is an aspect with regard to the application of this tetrazolium dye for the estimation of active bacteria in natural aquatic environments which hitherto has not been considered.