The MicroResp (TM) method allows soil respiration and microbial community physiological profiles to be determined colorimetrically in microplates. This method, however, neglects CO2 storage in the agar gel carrying the colorimetric indicator, and calcite dissolution associated with CO2-induced change in soil solution pH. Our objective was to improve the method by taking into account CO2 in the gel in the calculation of microbial respiration, describing the effect of microbial CO2 on the pH of the soil solution and calcite dissolution, and checking whether CO2 distribution among calcite, soil solution, air and gel is near equilibrium after incubation. We propose a thermodynamic equilibrium model describing (a) distribution of CO2 among calcite, soil solution, gel and air, (b) dissociations of water, carbonic acid, cresol red, and substrates in the gel and soil solution, (c) exchange of adsorbed cations with H3O+ in the gel, and (d) calcite dissolution in soil. In-gel experiments were designed to calibrate the model, quantify the rate of CO2 exchange with air, and compare conservation procedures. On-soil experiments were designed to check whether calcite dissolution is near equilibrium and whether the model predicts the effect of CO2 on the pH of the solution. In-microplate experiments were designed to assess the effects of incubation period and soil quantity on estimated microbial respiration. The model can describe the distribution and speciation of CO2 in the gel, the soil solution and the air space of each microplate well. Initial properties of the gel vary with storage: soda lime partly extracts CO2 supplied as NaHCO3, and dries out the gel, which can skew the calibration. When incubation is over, the proportion of microbial CO2 in the gel is higher at lower microbial respiration. Incubations shorter than 4 h underestimate microbial respiration due to the slow diffusion of CO2 in the gel. CO2 in the soil solution cannot be overlooked; it decreases the soil pH and may promote calcite dissolution in calcareous soil. It is important to precisely estimate initial CO2 air fraction and to control temperature, which affects both thermodynamic constants and microorganisms.