Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Dec;62(12):4587–4593. doi: 10.1128/aem.62.12.4587-4593.1996

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

S Ullrich, B Karrasch, H Hoppe, K Jeskulke, M Mehrens
PMCID: PMC1389009  PMID: 16535471

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 (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.

Full Text

The Full Text of this article is available as a PDF (205.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Coallier J., Prévost M., Rompré A., Duchesne D. The optimization and application of two direct viable count methods for bacteria in distributed drinking water. Can J Microbiol. 1994 Oct;40(10):830–836. doi: 10.1139/m94-132. [DOI] [PubMed] [Google Scholar]
  2. Dutton R. J., Bitton G., Koopman B. Malachite green-INT (MINT) method for determining active bacteria in sewage. Appl Environ Microbiol. 1983 Dec;46(6):1263–1267. doi: 10.1128/aem.46.6.1263-1267.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fuhrman J. A., Azam F. Bacterioplankton secondary production estimates for coastal waters of british columbia, antarctica, and california. Appl Environ Microbiol. 1980 Jun;39(6):1085–1095. doi: 10.1128/aem.39.6.1085-1095.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gahan P. B., Kalina M. The use of tetrazolium salts in the histochemical demonstration of succinic dehydrogenase activity in plant tissues. Histochemie. 1968;14(1):81–88. doi: 10.1007/BF00268035. [DOI] [PubMed] [Google Scholar]
  5. Hobbie J. E., Daley R. J., Jasper S. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol. 1977 May;33(5):1225–1228. doi: 10.1128/aem.33.5.1225-1228.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hurst C. J., Blannon J. C., Hardaway R. L., Jackson W. C. Differential Effect of Tetrazolium Dyes upon Bacteriophage Plaque Assay Titers. Appl Environ Microbiol. 1994 Sep;60(9):3462–3465. doi: 10.1128/aem.60.9.3462-3465.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. King L. K., Parker B. C. A simple, rapid method for enumerating total viable and metabolically active bacteria in groundwater. Appl Environ Microbiol. 1988 Jun;54(6):1630–1631. doi: 10.1128/aem.54.6.1630-1631.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kogure K., Simidu U., Taga N. A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol. 1979 Mar;25(3):415–420. doi: 10.1139/m79-063. [DOI] [PubMed] [Google Scholar]
  9. LESTER R. L., SMITH A. L. Studies on the electron transport system. 28. The mode of reduction of tetrazolium salts by beef heart mitochondria; role of coenzyme Q and other lipids. Biochim Biophys Acta. 1961 Mar 4;47:475–496. doi: 10.1016/0006-3002(61)90543-1. [DOI] [PubMed] [Google Scholar]
  10. Maki J. S., Remsen C. C. Comparison of two direct-count methods for determining metabolizing bacteria in freshwater. Appl Environ Microbiol. 1981 May;41(5):1132–1138. doi: 10.1128/aem.41.5.1132-1138.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Meyer-Reil L. A. Autoradiography and epifluorescence microscopy combined for the determination of number and spectrum of actively metabolizing bacteria in natural water. Appl Environ Microbiol. 1978 Sep;36(3):506–512. doi: 10.1128/aem.36.3.506-512.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Novitsky J. A., Morita R. Y. Survival of a psychrophilic marine Vibrio under long-term nutrient starvation. Appl Environ Microbiol. 1977 Mar;33(3):635–641. doi: 10.1128/aem.33.3.635-641.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pyle B. H., Broadaway S. C., McFeters G. A. A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water. Appl Environ Microbiol. 1995 Jul;61(7):2614–2619. doi: 10.1128/aem.61.7.2614-2619.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pyle B. H., Broadaway S. C., McFeters G. A. Factors affecting the determination of respiratory activity on the basis of cyanoditolyl tetrazolium chloride reduction with membrane filtration. Appl Environ Microbiol. 1995 Dec;61(12):4304–4309. doi: 10.1128/aem.61.12.4304-4309.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rodriguez G. G., Phipps D., Ishiguro K., Ridgway H. F. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl Environ Microbiol. 1992 Jun;58(6):1801–1808. doi: 10.1128/aem.58.6.1801-1808.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Roslev P., King G. M. Application of a tetrazolium salt with a water-soluble formazan as an indicator of viability in respiring bacteria. Appl Environ Microbiol. 1993 Sep;59(9):2891–2896. doi: 10.1128/aem.59.9.2891-2896.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schaule G., Flemming H. C., Ridgway H. F. Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water. Appl Environ Microbiol. 1993 Nov;59(11):3850–3857. doi: 10.1128/aem.59.11.3850-3857.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smith J. J., Howington J. P., McFeters G. A. Survival, physiological response and recovery of enteric bacteria exposed to a polar marine environment. Appl Environ Microbiol. 1994 Aug;60(8):2977–2984. doi: 10.1128/aem.60.8.2977-2984.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Smits J. D., Riemann B. Calculation of cell production from [h]thymidine incorporation with freshwater bacteria. Appl Environ Microbiol. 1988 Sep;54(9):2213–2219. doi: 10.1128/aem.54.9.2213-2219.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stellmach J. Fluorescent redox dyes. 1. Production of fluorescent formazan by unstimulated and phorbol ester- or digitonin-stimulated Ehrlich ascites tumor cells. Histochemistry. 1984;80(2):137–143. doi: 10.1007/BF00679987. [DOI] [PubMed] [Google Scholar]
  21. Stellmach J., Severin E. A fluorescent redox dye. Influence of several substrates and electron carriers on the tetrazolium salt-formazan reaction of Ehrlich ascites tumour cells. Histochem J. 1987 Jan;19(1):21–26. doi: 10.1007/BF01675289. [DOI] [PubMed] [Google Scholar]
  22. Tabor P. S., Neihof R. A. Improved method for determination of respiring individual microorganisms in natural waters. Appl Environ Microbiol. 1982 Jun;43(6):1249–1255. doi: 10.1128/aem.43.6.1249-1255.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thom S. M., Horobin R. W., Seidler E., Barer M. R. Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity. J Appl Bacteriol. 1993 Apr;74(4):433–443. doi: 10.1111/j.1365-2672.1993.tb05151.x. [DOI] [PubMed] [Google Scholar]
  24. Winding A., Binnerup S. J., Sørensen J. Viability of indigenous soil bacteria assayed by respiratory activity and growth. Appl Environ Microbiol. 1994 Aug;60(8):2869–2875. doi: 10.1128/aem.60.8.2869-2875.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yu F. P., McFeters G. A. Physiological responses of bacteria in biofilms to disinfection. Appl Environ Microbiol. 1994 Jul;60(7):2462–2466. doi: 10.1128/aem.60.7.2462-2466.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yu W., Dodds W. K., Banks M. K., Skalsky J., Strauss E. A. Optimal staining and sample storage time for direct microscopic enumeration of total and active bacteria in soil with two fluorescent dyes. Appl Environ Microbiol. 1995 Sep;61(9):3367–3372. doi: 10.1128/aem.61.9.3367-3372.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zimmermann R., Iturriaga R., Becker-Birck J. Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl Environ Microbiol. 1978 Dec;36(6):926–935. doi: 10.1128/aem.36.6.926-935.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES