CO 2 -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, %O ₂ -μR, in which the consumption of dissolved O ₂ is monitored with respect to incubation time, t . In %O ₂ -μR the 'bioreactor' often comprises a canonical plastic tube with a small %O ₂ 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, %CO ₂ -μR, in which the production of CO ₂ in the growth medium is monitored. In %CO ₂ -μR, the 'bioreactor' is the same as that used in %O ₂ -μR, but with a small 3D printed, colour-based %CO ₂ indicator set in its base and its apparent absorbance, A ', is measured at any t , as it is related to the %CO ₂ dissolved in the inoculated growth medium. Under aerobic conditions, different inoculations of the facultative anaerobe, E. coli , of different concentrations (10 ¹ -10 ⁸ 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 %CO ₂ -μR study, to those generated for the same system but using a commercial %O ₂ -μR system, is used to demonstrate method equivalence. A study of the same system, under anaerobic conditions, using %CO ₂ -μR, shows that %CO ₂ -μR is suitable for measuring the BL of anaerobes. The potential of %CO ₂ -μR for measuring the bacterial load of CO ₂ -generating aerobes and anaerobes is discussed briefly.