Enumeration of actively respiring bacteria in soil using the CTC-SYBR green I double-staining method

  • Ryuda Noriko
    Division of Soil Environment, Faculty of Agriculture, Saga University:The United Graduate School of Agricultural Sciences, Kagoshima University
  • Wang Xiaodan
    Division of Soil Environment, Faculty of Agriculture, Saga University
  • Ueno Daisuke
    Division of Soil Environment, Faculty of Agriculture, Saga University
  • Inoue Koichi
    Division of Soil Environment, Faculty of Agriculture, Saga University
  • Someya Takashi
    Division of Soil Environment, Faculty of Agriculture, Saga University

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Other Title
  • CTC-SYBR Green I二重蛍光染色法による土壌中の呼吸活性陽性菌の定量
  • CTC-SYBR Green 1二重蛍光染色法による土壌中の呼吸活性陽性菌の定量
  • CTC SYBR Green 1ニジュウ ケイコウ センショクホウ ニ ヨル ドジョウ チュウ ノ コキュウ カッセイ ヨウセイキン ノ テイリョウ

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Abstract

The tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been employed for the detection and enumeration of respiratory-active bacteria in environmental samples. In this method, CTC was reduced by a respiratory chain to form CTC-formazan crystals, which were deposited intracellularly and were enumerated under an epifluorescence microscope. We evaluated a CTC-SYBR Green I double-staining (CTC-SG) method, by which both CTC-formazan particles and whole cells could be observed by CTC and SG, respectively. Pure culture studies of several Gram-positive and -negative bacteria revealed that one or more CTC-formazan crystals were observed in a single bacterial cell and extracellularly. Consequently, the conventional CTC method gave counts 1.2-1.6 times higher than the CTC-SG method, suggesting overestimation by the CTC method. A starved culture of E coil showed the presence of viable but nonculturable cells, as detected by the CTC-SG method after two weeks of incubation. The counts of metabolically active bacteria in upland and paddy soils by the CTC-SG method were 4.5×10^8-1.4×10^9 cells g^<-1> dry soil, which were as much as 10-12% of the total bacterial count and 1.5-5.2 times higher than the plate counts. These results suggest that the CTC-SG method gives more accurate counts of respiratory-active bacteria in soil samples than the conventional CTC method.

Journal

  • Soil Microorganisms

    Soil Microorganisms 64 (1), 18-24, 2010

    Japanese Society of Soil Microbiology

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