CO2-Based micro-respirometry for measuring bacterial load under aerobic and anaerobic conditions†

The bacterial load (BL), or total viable count, of aerobes can be measured using micro-respirometry, %O₂-μR, in which the consumption of dissolved O₂ is monitored with respect to incubation time, t. In %O₂-μR the ‘bioreactor’ often comprises a canonical plastic tube with a small %O₂ sensor; it is simple, fast and accurate and used in automated, commercial instruments for measuring BL. Here we show that it is also possible to measure BL using a new form of micro-respirometry, %CO₂-μR, in which the production of CO₂ in the growth medium is monitored. In %CO₂-μR, the ‘bioreactor’ is the same as that used in %O₂-μR, but with a small 3D printed, colour-based %CO₂ indicator set in its base and its apparent absorbance, A′, is measured at any t, as it is related to the %CO₂ dissolved in the inoculated growth medium. Under aerobic conditions, different inoculations of the facultative anaerobe, E. coli, of different concentrations (10¹–10⁸ colony forming units (CFU) per mL) are used to generate a series of A′ vs. t profiles, and a straight-line calibration curve. Statistical comparative analysis of the results generated in the above %CO₂-μR study, to those generated for the same system but using a commercial %O₂-μR system, is used to demonstrate method equivalence. A study of the same system, under anaerobic conditions, using %CO₂-μR, shows that %CO₂-μR is suitable for measuring the BL of anaerobes. The potential of %CO₂-μR for measuring the bacterial load of CO₂-generating aerobes and anaerobes is discussed briefly.