Fluorescent assay for bacterial gram reaction

Abstract

The invention relates to a method of analyzing a sample thought to contain bacteria using an aqueous solution comprising one or more fluorescent dyes: a fluorescent dye of formula I, a fluorescent dye of formula II, a fluorescent dye of formula III, and a fluorescent dye of formula IV. Each of the dyes differ each from the other in their affinity for nucleic acids and in their spectral response to different types of bacteria in the sample. The first three dyes are nucleic acid stains and the fourth dye is a fluorescent reagent that binds selectively to cell surface components. The fluorescent dyes of formula I are highly membrane-permeant cyanine dye derivatives and label all bacteria, whether live or dead, whether Gram positive or Gram negative. The dyes of formula II label only live Gram positive bacteria and label all dead bacteria, whether Gram positive or negative. The dyes of formula II bind to nucleic acids preferentially with respect to the dyes of formula I. Fluorescent formula III dyes are membrane impermeant dyes that give a fluorescent signal only in cells with compromised plasma membrane integrity, whether Gram negative or Gram positive, and have a much higher binding affinity for nucleic acids than the fluorescent dyes of either formula I or formula II. Formula IV fluorescent dyes preferentially bind to an exterior component of a bacterium. The dyes are combined with a sample suspected of containing bacteria and illuminated at an appropriate wavelength to differentiate, according to the fluorescence response, live Gram negative, dead Gram negative, live Gram positive and dead Gram positive bacteria in the sample.