Effect of assay conditions on the measurement of dehydrogenase activity of Streptomyces venezuelae using triphenyl tetrazolium chloride


Jadomycin is an antibiotic that has shown activities against bacteria, yeasts and fungi as well as cytotoxic properties to cancer cells. Because of the wide range of its inhibitory actions, jadomycin shows promise as a novel antibiotic and cancer treatment drug. Streptomyces venezuelae are aerobic bacteria that produce jadomycin and the size of bacterial population can significantly affect the yield of jadomycin. Therefore, the bacterial population must be accurately measured in order to standardize the reproducibility of jadomycin production process. In this study, a dehydrogenase activity measurement test, using triphenyl tetrazolium chloride (TTC), was used to measure the dehydrogenase activity of Streptomyces venezuelae during growth in maltose-yeast extract-malt extract (MYM) broth. The aims were to evaluate the effectiveness of the test for measuring microbial growth and to study the effects of the test conditions (incubation time, incubation temperature and medium pH) on triphenyl formazan (TF) yield. The results showed that the TF yield was highly correlated to the optical density. The highest TF yield was observed at a pH of 6 at all incubation times and temperature. Lower TF yields were obtained at higher temperature (40 and 50oC) compared to those obtained at lower temperatures (22 and 30oC). The difference between the yields obtained at 22oC and 30oC were not significant. The differences between incubation time were also not significant. The recommended test conditions are an incubation time of 1 hour at a temperature of 30oC and a pH of 6 followed by three extractions using methanol.

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Burdock, T. , Brooks, M. , Ghaly, A. and Dave, D. (2011) Effect of assay conditions on the measurement of dehydrogenase activity of Streptomyces venezuelae using triphenyl tetrazolium chloride. Advances in Bioscience and Biotechnology, 2, 214-225. doi: 10.4236/abb.2011.24032.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Jakeman, D.L., Farrell, S., Young, W., Doucet, R.J. and Timmons SC (2005) Novel and expanded jadomycins incorporating non-proteogenic amino acids. Bioorganic and Medicinal Chemistry Letters, 15(23), 5280-5283. doi:10.1016/j.bmcl.2005.08.047
[2] Zheng, J. Rix, U., Mattingly, C., Adams, V., Chen, Q., Rohr, J. and Yang, K. (2005) Cytotoxic activities of new jadomycin derivatives. Journal of Antibiotics, 58(6), 405-408. doi:10.1038/ja.2005.51 J
[3] Burdock, T.J., Giffin, A.H., Brooks, M.S. and Ghaly, A.E. (2008) Heat balance analysis during the production of jadomycin C. American Journal of Biochemistry and Biotechnology, 4(1), 7-18. ISSN: 1553-3468
[4] Doull, J.L., Singh, A.K., Hoare, M. and Ayer, S.W. (1994) Conditions for the production of jadomycin B by Streptomyces venezuelae ISP5230: effects of heat shock, ethanol treatment and phage infection. Journal of Industrial Microbiology, 13(2), 120-125. doi:10.1007/BF01584109
[5] Jakeman, D.L., Bandi, S., Graham, C.L., Reid, T.R., Wentzell, J.R. and Douglas, S.E. (2009) Antimicrobial activities of jadomycin B and structurally related analogues. Antimicrobial Agents and Chemotherapy, 53(3), 1245-1247. doi:10.1128/AAC.00801-08
[6] Jakeman, D.L., Graham, C.L., Young, W. and Vining, L.C. (2006) Culture conditions improving the production of jadomycin B. Journal of Industrial Microbiology and Biotechnology, 33, 767-772. doi:10.1007/s10295-006-0113-4
[7] Friedel, J.K., Molter, K. and Fischer, W.R. (1994) Comparison and improvement of methods for determining soil dehydrogenase activity by using triphenyltetrazolium chloride and iodonitrotetrazolium chloride. Biology and Fertility of Soils, 18, 291-296. doi: 10.1007/BF00570631
[8] Xie, J., Hu, W., Pei, H., Dun, M. and Qi, F. (2008) Detection of amount and activity of living algae in fresh water by dehydrogenase activity (DHA). Environmental Monitoring and Assessment, 146, 473-478. doi: 10.1007/s10661-008-0250-5
[9] Chander, K. and Brookes, P.C. (1991) Is the dehydrogenase assay invalid as a method to estimate microbial activity in copper contaminated soils? Soil Biology and Biochemistry, 23, 909-915. doi:10.1016/0038-0717(91)90170-O
[10] Rogers, J.E. and Li, S.W. (1985) Effect of metals and other inorganic ions on soil microbial activity: Soil dehydrogenase assay as a simple toxicity test. Bulletin Environmental Contamination and Toxicology, 34, 858-865. doi:10.1007/BF01609817
[11] Yin, J., Liu, X. and Song, X. (2001) A method of extraction at normal temperature for measurement of TTC-dehydrogenase activity and its application. China Water and Wastewater, 17(8), 23-25.
[12] Beloti, V., Barros, M., de Freitas, J.C., Nero, L.A., de Souza, J.A., Santana, E.H.W. and Franco, B.D.G.M. (1998) Frequency of 2,3,5-triphenyltetrazolium chloride (TTC) non-reducing bacteria in pasteurized milk. Revista de Microbiologia, 30,137-140. doi:10.1590/S0001-37141999000200009
[13] Mahmoud, N.S. and Ghaly, A.E. (2004) Influence of temperature and pH on the nonenzymatic reduction of triphenyltetrazolium chloride. Biotechnology Progress, 20, 346-353. doi: 10.1021/bp030029h
[14] Tarafdar, P.J.C. (2003) 2,3,5-Triphenyltetrazolium chloride (TTC) as electron acceptor of culturable soil bacteria, fungi and actinomycetes. Biology and Fertility of Soils, 38, 186–189. doi: 10.1007/s00374-003-0600-y
[15] Ohara, M.T. and Saito, T. (1995) Application of triphenyltetrazolium chloride in microbial limit test of pharmaceuticals and cosmetics. Journal of AOAC International, 78(6), 1524-1529. ISSN: 0006-6648
[16] Mahmoud, N.S. (2005) Novel Biotechnological Approach for the Production of chitin and de-icing agents. Ph.D Thesis, Dalhousie University, Halifax, Canada.
[17] Burdock, T.J. (2009) Development of a dehydrogenase activity measurement test for streptomyces venezuelae using triphenyl tetrazolium chloride. MASc. Thesis, Dalhousie University, Halifax, Canada.
[18] Ghaly, A.E., Kok, R., Ingrahm, J.M. (1989). Growth rate determination of heterogeneous microbial population in swine manure. Applied Biochemistry and Biotechnology, 22, 59-78. doi: 10.1007/BF02922697
[19] Abdel-Fattah, Y.R. (2007) Application of fractional factorial design for the development of production media for the pikromycin macrolide family by Streptomyces venezuelae. Trends in Applied Science Research, 2, 472-482. doi: 10.3923/tasr.2007.472.482
[20] Glazebrook, M, Doull, J.L., Stuttardan, C. and Vining, L.C. (1990) Sporulation of Streptomyces venezuelae in submerged cultures. Journal of General Microbiology, 136, 581-588. Retrieved on 23rd November, 2011 from Hhttp://mic.sgmjournals.org/cgi/reprint/136/3/581.pdf
[21] Backor, M. and Fahselt, D. (2005) Tetrazolium reduction as an indicator of environmental stress in lichens and isolated bionts. Environmental Experimental Botany, 53(2), 125-133. doi:10.1016/j.envexpbot.2004.03.007
[22] Jones, P.H. and Prasad, D, (1969) The use of tetrazolium salts as a measure of sludge activity. Water Pollution Control Federation, 41(11), R441-R449. Retrieved on 23rd November, 2011 from Hhttp://www.jstor.org/stable/25039175
[23] Mersi, W.V. and Sehinne, F. (1991) An improved and accurate method for determining the dehydrogenase activity of soils with iodonitrotetrazolium chloride. Biology and Fertility of Soils, 11, 216-220. doi: 10.1007/BF00335770
[24] Brzezińska, M., St?pniewska, Z. and St?pniewski, W. (2001) Dehydrogenase and catalase activity of soil irrigated with municipal wastewater. Polish Journal of Environmental Studies, 10, 307-311. Retrieved on 15th February 2011 from Hhttp://6csnfn.pjoes.com/pdf/10.5/307-311.pdf
[25] Ghaly, A.E. and Ben-Hassan, R.M. (1993) Dehydrogenase activity measurement in yeast fermentation. Applied Biochemistry Biotechnology, 43(2), 77-91. doi:10.1007/BF02916434
[26] Ghaly, A.E. and Mahmoud, N.S. (2006) Optimum conditions for measuring dehydrogenase activity of Aspergillus niger using TTC. American Journal of Biochemistry and Biotechnology, 2,186-194. ISSN: 1553-3468
[27] Mahmoud, N.S. and Ghaly, A.E. (2006) Optimum condition for measuring dehydrogenase activity of Aspergillus niger using TTC. American Journal of Biochemistry and Biotechnology, 2(4), 186-194. ISSN 1553-3468
[28] Mathew, M. and Obbard, J.P. (2001) Optimization of dehydrogenase activity measurements in beach sediments contaminated with petroleum hydrocarbons. Biotechnology Letters, 23, 227-230. DOI: 10.1023/A:1005613406948
[29] Tengerdy, R.P., Nagy, J.G. and Martin, B, (1967) Quantitative measurement of bacterial growth by the reduction of tetrazolium salts. Applied Microbiology, 15(4), 954-955. Retrieved on 15th February 20111 from Hhttp://aem.asm.org/cgi/reprint/15/4/954H.
[30] Griebe, T., Schaule, G. and Wuertz, S. (1997) Determination of microbial respiratory and redox activity in activated sludge. Journal of Industrial Microbiology and Biotechnology, 19, 118-122. doi: 10.1038/sj.jim.2900431
[31] Trevors, J.T. (1984) Effect of substrate concentration, inorganic nitrogen, O2 concentration, temperature and pH on dehydrogenase activity in soil. Plant and Soil, 77, 285-293. doi:10.1007/BF02182931.
[32] Tiquia, S.M., Wan, J.H.C. and Tam, N.F.Y. (2002) Dynamics of yard trimmings composting as determined by dehydrogenase activity. ATP content, arginine ammonification, and nitrification potential. Process Biochemistry, 37, 1057-1065. doi:10.1016/S0032-9592(01)00317-X
[33] Zhao, X., Li, H., Wu, Q., Li, Y., Zhao, C. and Zhang, Y. (2010) Analysis of dehydrogenase activity in phytoremediation of composite pollution sediment. 2nd Conference on Environmental Science and Information Application Technology. (ESIAT 2010), Wuhan, China. Retrieved on 7th April, 2011 from Hhttp://ieeexplore.ieee.org/stamp/stamp.jsp?arnumber=05567388
[34] Breed, R.S. (1957) Bergey’s Manual of Determinative Bacteriology, 7th Edition. Williams and Wilkins Company, Baltimore, Maryland.
[35] Doull, J.L., Ayer, S.W., Singh, A.K. and Thibault, P. (1993) Production of a novel polyketide antibiotic, jadomycin B, by Streptomyces venezuelae following heat-shock. Journal of Antibiotics, 46, 869-871. ISSN: 0021-8820.

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