Use of n-phenylmethazonium methosulphate oxidation of nadh in the quantification of oxygen uptake by oral bacteria under open-system conditions

Technically, open systems are more suited than closed systems for the measurement of oxygen uptake by bacteria present at high cell densities as in dental plaque. Moreover, measurements are easier to perform and can be done faster and continuously. However, quantification is more difficult and, except for steady-state conditions, has been semiquantitative. The stoichiometric oxidation of NADH by N-phenylmethazonium methosulphate (PMS) is a reaction used here to develop a formula that overcomes this problem and makes it possible to relate quantitatively the pO2 measured easily with an oxygen electrode to the amount of oxygen utilized by respiring bacteria provided with a given amount of substrate. The approach and formula consist of the summing of (i) the oxygen decrease observed during the period of bacterial oxygen consumption and (ii) the oxygen simultaneously entering from the atmosphere. To quantify the entry component, the rate of oxygen diffusion into the mixture needed to be determined: this was done by reducing or exhausting the oxygen content of the medium in various ways and determining the rate constant of atmospheric oxygen entry. The relation between the logarithm of the oxygen concentration (determined from pO2 values using the PMS-NADH reaction) and point of time in the oxygen resaturation process proved to be linear. This enabled calculation of a diffusion rate constant, k, for use in the oxygen utilization formula. The validity of this method for quantification of oxygen use was tested by comparing oxygen consumption determined from the pO2 tracing and the derived formula to the oxygen consumed when fixed amounts of NADH are oxidized by PMS. When oxygen uptake/substrate utilization molar ratios were calculated for each of several NADH concentrations, the resulting values were almost identical. The method proved reliable over most of the oxygen concentration range possible in this system.