Biology and Fertility of Soils

, Volume 11, Issue 3, pp 216–220

An improved and accurate method for determining the dehydrogenase activity of soils with iodonitrotetrazolium chloride

  • W. von Mersi
  • F. Schinner


Conditions for a rapid, precise [100 μg iodonitrotetrazolium chloride (INT)-formazan ml-1 assay mixture], and easily reproducible assay of potential soil dehydrogenase activity are described, using 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (iodonitrotetrazolium chloride, INT) as the substrate. Reduced iodonitrotetrazolium formazan (INTF) was measured by spectrophotometry (464 nm) after extraction with N,N-dimethylformamide and ethanol. With this method, the coloured complex formed is highly stable. The effects of pH, buffer concentration, temperature, substrate concentration, amount of soil weight, and reaction time on dehydrogenase activity were investigated. The rate of substrate hydrolysis was proportional to soil weight; the optimal INT reduction was achieved with 1 M TRIS buffer (pH 7.0) at 40 °C. It was possible to determine the biotic and abiotic substrate reduction by comparing assays of autoclaved and unsterile soil samples. Different investigations have confirmed that the intracellular enzyme is highly correlated with the microbial biomass, and indicate that this activity is suitable as an indirect parameter of microbial biomass, measurement.

Key words

Dehydrogenase activity Iodonitrotetrazolium chloride (INT) Microbial biomass Microbial activity 


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  1. Benefield CB, Howard PJA, Howard DM (1977) The estimation of dehydrogenase activity in soil. Soil Biol Biochem 9:67–70Google Scholar
  2. Bremner JM, Tabatabai MA (1973) Effects of some inorganic substances on TTC assy of dehydrogenase activity in soils. Soil Biol Biochem 5:385–386Google Scholar
  3. Casida LE Jr (1977) Microbial metabolic activity in soil as measured by dehydrogenase determinations. Appl Environ Microbiol 34:630–636Google Scholar
  4. Casida LE Jr, Klein DA, Santoro T (1964) Soil dehydrogenase activity. Soil Sci 98:371–376Google Scholar
  5. Chendrayan K, Adhya TK, Sethunathan N (1980) Dehydrogenase and invertase activities of flooded soils. Soil Biol Biochem 12:271–273Google Scholar
  6. Curl H Jr, Sandberg J (1961) The measurement of dehydrogenase activity in marine organisms. J Mar Res 19:123–138Google Scholar
  7. Glathe H, Thalmann H (1970) Über die mikrobielle Aktivität und ihre Beziehungen zu Fruchtbarkeitsmerkmalen einiger Ackerböden unter besonderer Berücksichtigung der Dehydrogenaseaktivität (TTC-Reduktion). 2. Mitt Zentralbl Bakteriol Parasitenkd, Infektionskr Hyg Abt 2 124:24–36Google Scholar
  8. Griffiths BS (1989) Improved extraction of iodonitrotetrazolium-formazan from soil with dimethylformamide. Soil Biol Biochem 21:179–180Google Scholar
  9. Klein Da, Loh TC, Goulding RL (1971) A rapid procedure to evaluate the dehydrogenase activity of soils low in organic matter. Soil Biol Biochem 3:385–387Google Scholar
  10. Lenhard G (1956) Die Dehydrogenaseaktivität des Bodens als Maß für die Mikroorganismentätigkeit im Boden. Z Pflanzenernaehr Bodenkd 73:1–11Google Scholar
  11. Ross DJ (1971) Some factors influencing the estimation of dehydrogenase activities of some soils under pasture. Soil Biol Biochem 3:97–110Google Scholar
  12. Skujins J (1973) Dehydrogenase: An indicator of biological activities in arid soils. Bull Ecol Res Commun (Stockholm) 17:235–241Google Scholar
  13. Stevenson IL (1959) Dehydrogenase activity in soils. Can J Microbiol 5:229–235Google Scholar
  14. Tabatabai MA (1982) Soil enzymes. In: Page AL, Miller RH, Keeney DR (eds) Methods of soil analysis: II. Chemical and microbiological propeties, 2nd edn. Am Soc Agron, Agronomy 10, Madison, Wisconsin, pp 903–947Google Scholar
  15. Thalmann A (1968) Zur Methodik der Bestimmung der Dehydrogenaseaktivität im Boden mittels Triphenyltetrazoliumchlorid (TTC). Landwirtsch Forsch 21:249–258Google Scholar
  16. Trevors JT (1984a) Effect of substrate concentration, inorganic nitrogen, O2 concentration, temperature and pH on dehydrogenase activity in soil. Plant and Soil 77:285–293Google Scholar
  17. Trevors JT (1984b) Dehydrogenase activity in soil: A comparison between the INT and TTC assay. Soil Biol Biochem 16:673–674Google Scholar
  18. Trevors JT (1986) Electron transport system activity in soil, sediment, and pure cultures. Crit Rev Microbiol 11:83–99Google Scholar
  19. Trevors JT, Mayfield CI, Inniss WE (1982) Measurement of electron transport system (ETS) activity in soil. Microb Ecol 8:163–168Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • W. von Mersi
    • 1
  • F. Schinner
    • 1
  1. 1.Institute of MicrobiologyUniversity of InnsbruckInnsbruckAustria

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