Aim: The aim of this work was to develop a tool to investigate the influence of soil factors on carbon utilization activity of single micro-organisms. Methods and Results: The assay for Rhizobium leguminosarum bv. trifolii in c-irradiated soil, using the MicroResp TM system, was optimized for sterility, incubation time, and moisture level. The optimized method was validated with experiments that assessed (i) differences in C utilization of different rhizobia strains and (ii) how this was affected by soil type. Carbon utilization differed among strains of the same species (and symbiovar), but some strains were more responsive to the soil environment than others. Conclusions: This novel modification of the MicroResp TM has enabled the scope of carbon-utilization patterns of single strains of bacteria, such as Rh. leguminosarum bv. trifolii, to be studied in soil. Significance and Impact of the Study: The system is a new tool with applications in microbial ecology adaptable to the study of many culturable bacterial and fungal soil-borne taxa. It will allow measurement of a microorganism's ability to utilize common C sources released in rhizosphere exudates to be measured in a physical soil background. This knowledge may improve selection efficiency and deployment of commercial microbial inoculants.