Patent ID: 7582483

Claim:
A method of detecting viable cells comprising steps of: (1) fluorescently staining cells in a sample by adding a fluorescent dye to the sample or putting the sample in contact with the fluorescent dye; (2) adding a quenching dye capable of absorbing fluorescence of the fluorescent dye to the sample that is stained with the fluorescent dye, or putting the sample in contact with the quenching dye; and (3) illuminating the sample stained with the fluorescent dye and the quenching dye with excitation light for the fluorescent dye, and collecting and detecting fluorescence emitted from the sample; wherein, in step (2), (a) the quenching dye is permeable through a membrane of a viable cell, (b) the quenching dye does not readily absorb the fluorescence of the fluorescent dye at a pH to be found in the viable cells, and (c) the quenching dye absorbs the fluorescence of the fluorescent dye at a pH substantially different from the pH in the viable cells, and the quenching dye is added to the sample or the sample is put in contact with the quenching dye under a pH that is substantially different from the pH in the viable cells; and in step (3), the sample stained with the fluorescent dye and the quenching dye is maintained at a pH substantially different from the pH in the viable cells.