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.
