A laboratory experiment was conducted to evaluate the effect of triphenyltetrazolium chloride (TTC) on soil microorganisms and the availability of pH characterization medium in BIOLOG plates. Application of TTC decreased the color development sharply and resulted in a great biocidal effect on the growth and reproduction of soil microorganisms, indicating that TTC can affect the discrimination on soil microbial community. The microtitration plates with 21 carbon sources and two different pH levels (4.7 and 7.0) were used to determine microbial community structure of eight red soils. The average utilization (average well colour development) of the carbon sources in the plates with different pH levels generally followed the same sigmoidal pattern as that in the traditional BIOLOG plates, but the pH 4.7 plates increased the discrimination of this technique, compared with the pH 7.0 plates. Since most tested soils are acid, it seemed that it’s better to use a suitable pH characterization medium for a specific soil in the sole carbon source test. A laboratory experiment was conducted to evaluate the effect of triphenyltetrazolium chloride (TTC) on soil microorganisms and the availability of pH characterization medium in BIOLOG plates. Application of TTC decreased the color development sharply and resulted in a great biocidal effect on the growth and reproduction of soil microorganisms, indicating that TTC can affect the discrimination on soil microbial community. The microtitration plates with 21 carbon sources and two different pH levels (4.7 and 7.0) were used to determine microbial community structure of eight red soils. color development in the plates with different pH levels generally followed the same sigmoidal pattern as that in the traditional BIOLOG plates, but the pH 4.7 plates increased the discrimination of this technique, compared with the pH 7.0 plates. soils are acid, it was that it’s better to use a suitable pH characterization me dium for a specific soil in the sole carbon source test.