Abstract

The present study focuses on the improvement of limitations of the bioluminescent ATP assay. An attempt was made to improve on the following: non-selective detection, limited cell population density quantification, and to distinguish between bioluminescent noises data and cell ATP, as a suitable technique for coliforms and Escherichia coli detection, as well as other cell strains. Results were achieved by combining growth kinetics of the cell population exponential phase, in 2% brilliant green lactose bile broth with bioluminescent assay, based on the reaction catalyzed by firefly luciferase. The equations applied to the description of kinetics on exponential growth phase were able to estimate the relative light units (RLU₀) corresponding to initial adenosine triphosphate (ATP₀) concentration. The developed equations also help to distinguish between background RLU and RLU₀ corresponding to cell ATP, and to analyze the kinetics with and without lag period in order to estimate RLU₀. The results demonstrated a good correlation with data obtained by standard methods for coliforms detection. The quantification of E. coli and coliforms could be performed within a period of 6 h at 3.24 x 10⁵ and 1.12 x 10³ CFU/mL from the calculation of initial ATP concentrations at 19 and 0.067 pmol/mL, respectively. Monitoring of growth kinetic parameters along with the bioluminescent detection of microorganisms in the selective medium during shorter test time has potential for their selective and sensitive quantification. The mathematical approach may be extended to the analysis of experimental data obtained by other techniques based on the cell metabolite detection.

Key words: Bioluminescence, coliforms, Escherichia coli, exponential phase of growth, adenosine triphosphate (ATP) detection.