Bioluminescent assay of total bacterial contamination of drinking water

Abstract

A bioluminescent assay of total bacterial contamination (TBC) of drinking water (DW) with a detection limit of approximately 1 CFU/mL and duration of less than 7 h has been developed. The protocol of the TBC assay comprises: incubation of water sample in nutrition broth supplemented with salts mixture, up to 6 h; filtration of bacterial suspension obtained through membrane filter (pore size 0.45 microm); release of bacterial ATP by dimethyl sulphoxide; determination of bacterial ATP concentration using highly sensitive ATP reagent based on recombinant Luciola mingrelica luciferase. To simplify the assay, special luminometer microcuvette Filtravette (New Horizons Diagnostics Corp., USA) are used. A good correlation (R=0.98) between ATP concentration measured after 6 h incubation and initial bacterial titre in DW was observed. Semi-quantitative TBC assay of DW is also available. The TBC value in DW is assessed by the fixation of incubation time required to detect a measurable bioluminescent signal: 3, 4 and 6 h corresponds to 100-1000, 10-100 and 1-10 CFU/mL, respectively.