BACTERIAL VIABILITY AND ANTIBIOTIC SUSCEPTIBILITY TESTING WITH SYTOX GREEN NUCLEIC-ACID STAIN

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with th ree positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids r esulted in a >500-fold enhancement in fluorescence emission (absorptio n and emission maxima at 502 and 523 nm, respectively), rendering bact eria with compromised plasma membranes brightly green fluorescent. SYT OX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labe led with SYTOX Green stain was typically >10-fold brighter than that f rom intact organisms. Bacterial suspensions labeled with SYTOX Green s tain emitted green fluorescence in proportion to the fraction of perme abilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric appro aches were used to quantify the effect of beta-lactam antibiotics on t he cell membrane integrity of Escherichia coli. Detection and discrimi nation of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodid e-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for m easuring bacterial viability and antibiotic susceptibility.