System for detecting growth in microorganisms

Abstract

A system for identifying within a mixed population, a group of microorganisms replicating in a culture medium specific to that group, which culture medium includes a fluorescence inhibitor which the group incorporates in their nucleic acid upon replication. In one embodiment, a sample of the microorganism population is dyed with two different fluorescent dyes which are specific to nucleic acid, the fluorescent emissivity of one of the dyes being reduced or quenched by the presence of the inhibitor in the dyed nucleic acid, the fluorescent emissivity of the other of the dyes being unaffected by the presence of the inhibitor. The ratio of intensities of the fluorescent emission from the two dyes is independent of the total nucleic acid content of each microorganism, but is dependent upon the extent of incorporation of the inhibitor into the nucleic acid, so serves as a marker or identifier of a replicated organism. In another embodiment of the invention, the sample of the population is dyed with but the one quenchable dye and the dyed organisms are exposed to high intensity radiation which very rapidly bleaches the dye. From the bleaching characteristics such as the time required for the fluorescent emission to decay from its initial intensity to l/e, or from the ratio of the initial intensity of emission to the integrated emission during bleaching, one can determine independently of the total amount of nucleic acid, changes in the quantum efficiency of the dye caused by the incorporation of the inhibitor in the nucleic acid, thereby identifying replicating organisms.