The MicroResp™ method allows soil respiration and microbial community physiological profiles to be determined colorimetrically in microplates. This method, however, neglects CO 2 storage in the agar gel carrying the colorimetric indicator, and calcite dissolution associated with CO 2 -induced change in soil solution pH. Our objective was to improve the method by taking into account CO 2 in the gel in the calculation of microbial respiration, describing the effect of microbial CO 2 on the pH of the soil solution and calcite dissolution, and checking whether CO 2 distribution among calcite, soil solution, air and gel is near equilibrium after incubation. We propose a thermodynamic equilibrium model describing (a) distribution of CO 2 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 H 3 O + in the gel, and (d) calcite dissolution in soil. In-gel experiments were designed to calibrate the model, quantify the rate of CO 2 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 CO 2 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 CO 2 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 CO 2 supplied as NaHCO 3 , and dries out the gel, which can skew the calibration. When incubation is over, the proportion of microbial CO 2 in the gel is higher at lower microbial respiration. Incubations shorter than 4h underestimate microbial respiration due to the slow diffusion of CO 2 in the gel. CO 2 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 CO 2 air fraction and to control temperature, which affects both thermodynamic constants and microorganisms.