Bl. Roth et al., BACTERIAL VIABILITY AND ANTIBIOTIC SUSCEPTIBILITY TESTING WITH SYTOX GREEN NUCLEIC-ACID STAIN, Applied and environmental microbiology, 63(6), 1997, pp. 2421-2431
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.