CO2-sensitive inks for the rapid measurement of total viable count (TVC) using micro-respirometry†

At present, micro-respirometry for measuring total viable count, O₂ μR-TVC, is based on the time taken, TT, for an inoculum to significantly reduce the dissolved O₂ level (typically from 21% to ≤ 10.5%). Here, a simple kinetic model relevant to μR-TVC is presented which describes the growth of the bacteria from an initial inoculum, N_o, to a maximum level, N_max, and concomitant consumption of O₂ and generation of CO₂, in which the half-way time point, , corresponds to N_max/N_o = 0.5, at which point %O₂ = %CO₂ = 10.5%. The model shows that it is not possible to reduce the TT in O₂ μR-TVC below , as TT increases above with increasing sensitivity of the O₂ sensor. In contrast, the same model shows that if a CO₂ sensor is used instead, TT can be reduced significantly below and consequently CO₂ μR-TVC could be made much faster than conventional O₂ μR-TVC. To test this model prediction, a range of colourimetric CO₂ sensors of varying sensitivity, α, were prepared and used to make CO₂ μR-TVC measurements. The results confirm that the greater the sensitivity of the sensor, the shorter the TT, as predicted by the kinetic model. Two CO₂ indicators, one of moderate sensitivity and one of high sensitivity were used to generate straight-line log(CFU mL⁻¹) vs. TT calibration plots, which can then be used to determine the unknown TVCs of subsequent samples. The future of CO₂ μR-TVC as a possible new, faster alternative to conventional O₂ μR-TVC is discussed briefly.