Ammonia colourimetric indicator for measuring urease and ureolytic bacteria concentrations

An effectively irreversible colourimetric ammonia (NH₃) indicator is used to develop two new methods for determining the concentrations of, (i) urease [urease], and (ii) ureolytic bacteria under aerobic and anaerobic conditions. In both methods digital colour analysis (DCA) of a photographic image of the indicator is used to provide a value for the apparent absorbance, A', which reflects the level of NH₃ present. In (i) the indicator is placed in a solution containing urea and the urease sample under test, and photographed as a function of incubation time, t. DCA of the photographs yields an A' vs t plot, from which a value for the time taken for the indicator to reach its half-way colour changing point, t₅₀, is determined, the reciprocal of which is directly proportional to [urease]. In (ii), the indicator is placed in a Falcon™ tube containing a urea-based growth medium inoculated with a ureolytic bacterium of known concentration (units: CFU/mL), and photographed as a function of t. The resulting 'S' shaped A' vs t profile is used to determine a value of the half-way colour change, the threshold time, TT, which is shown to be directly proportional to log(CFU/mL) over the range 10¹-10⁸ CFU/mL for ureolytic bacteria such as P. stuartii and P. mirabilis, even in the presence of a non-ureolytic bacterial species, such as E. coli. The latter novel NH₃ indicator based micro-respirometry method for measuring TVC (NH₃ μR-TVC) works under anaerobic and aerobic conditions.