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.