ADENOSINE TRIPHOSPHATE ENHANCES THE FLUORESCENCE OF 4′,6‐DIAMIDINO‐2‐PHENYLINDOLE (DAPI)‐LABELED ESCHERICHIA COLI O157:H71

Abstract

ABSTRACT Live cells of E. coli O157:H7 were labeled by 4′,6‐diamidino‐2‐phenylindole (DAPI) in buffers of different pH. The extent of labeling was relatively insensitive to pH in the range of 6.5 to 9.5. The fluorescence intensity of ± 104 DAPI‐labeled bacteria per mL in optical cuvettes could be detected by a luminescence spectrometer. With a fluorescence microplate reader attachment, less than 103 of labeled bacteria could be measured. DAPI‐labeling inhibited the growth and respiratory activities of the bacteria. The addition of 0.5 to 6 mM concentrations of ATP induced a substantial increase in the fluorescence of labeled bacteria. Maximal enhancement by ATP was observed from bacteria still maintaining low levels of physiological activities. The enhancement favored more alkaline media with pH greater than 9. A replacement of ATP with ADP or AMP diminished the extent of enhancement. Other triphosphate nucleotides did not enhance fluorescence of DAPI‐labeled bacteria. Comparable ATP enhancements were also observed with Pseudomonas alcaligenes and Shewanella putrefaciens. Solubilization/destruction of cell membranes of labeled bacteria by detergents essentially eliminated the ATP enhancement. Absorption and fluorescence spectroscopic measurements indicated that ATP could interact with free and bound DAPI. These results suggest that observed ATP enhancement in fluorescence intensity of DAPI labels in intact cells may be applied to increase the sensitivity of microorganism detection.