Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride

Abstract

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 14 C-labeled glucose), and [ 14 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 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.

Evaluation of cell activity and of methods for the cultivation of bacteria from a natural lake community

“…Further support for this conclusion comes from the low fraction of CTC-reducing cells that has also been reported for bacteria from drinking water samples or from soil [34,37]. One major reason for these low counts of CTC-reduc-ing bacteria may be the toxicity of CTC which inhibits glucose uptake and respiration, thymidine incorporation, and growth on agar plates [3].…”

“…Generally less than 1% of the total number of bacterial cells which are present in natural seawater, soil, or freshwater samples can be cultured by standard laboratory methods [1^3]. However, up to 50% [4] and in some instances even 90% [5] of the bacterial cells appear to be metabolically active when assessed by the direct viable count technique [6], by microautoradiography with radiolabelled substrates [3,4,7], or by monitoring the presence of active electron transport systems in the cells with tetrazolium 0168-6496 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.…”

Application of laser scanning for the rapid and automated detection of bacteria in water samples

“…Such problems have not been seen with Gram-negative organisms such as Escherichia coli where no significant difference has been observed between the CB assay, CTC assay and plate counts. In a recent review, Ullrich et al (1996) describe a growing body of evidence which suggests that CTC is unsuitable for the enumeration of bacteria in environmental samples. The possible expla-nations for obtaining lower counts by CTC than by other direct methods of enumeration were (i) not all bacteria are capable of reducing tetrazolium salts (Thom et al 1993), (ii) the assay is not sensitive enough to detect bacteria with low rates of metabolic activity (Gasol et al 1995) and (iii) although CTC has been assumed to be non-toxic to bacteria (Rodriguez et al 1992), it has been reported that bacterial metabolic activity can be directly suppressed/inhibited by CTC, causing an under-estimation of the total number of bacteria within a sample (Ullrich et al 1996).…”

“…Another widely applied technique for the direct detection of viable bacteria uses the redox dye 5-cyano-2,3,4-ditolyl tetrazolium chloride (CTC) (Winding et al 1994;Oliver et al 1995;Ullrich et al 1996). This is a tetrazolium salt which is reduced by the respiratory electron transport chain to insoluble, fluorescent, formazan crystals which accumulate within individual cells.…”

Enumeration and characterization of bacteria in mineral water by improved direct viable count method

“…All these method present drawbacks; DAPI binds to numerous macromolecules (Kepner and Pratt 1994), Gramnegative isolates are impermeable to FDA (Tsuji et al 1995) and rhodamine 123 (Kaprelyants and Kell 1992), and CTC has a toxic effect on bacterial metabolism (Ullrich et al 1996).…”