TABLE 1 .

Substrates commonly used to measure direct efflux

Substrate	Details	Advantage(s)	Disadvantage(s)	Reference(s)
Ethidium bromide	DNA-intercalating dye; fluoresces when bound to DNA; excitation wavelength, 530 nm; emission wavelength, 600 nm	Well-validated substrate of many efflux pumps such as the RND pump AcrB	Concentrates in cytoplasm, so efflux is slow because there must be a dissociation step and probably more than one efflux event; likely to underestimate efflux level	9, 41
Nile Red	Periplasmic; lipophilic dye that binds to membrane phospholipids; fluoresces weakly in aqueous solutions but strongly fluorescent in nonpolar environments such as the membrane; excitation wavelength, 552 nm; emission wavelength, 636 nm; assay uses stationary-phase cells	Periplasmic, so good for studying RND efflux pumps such as AcrB; long maximum emission wavelength (636 nm) means that there is less of a problem with interference when measuring competition with other substrates; better signal-to-noise ratio than ethidium bromide; efflux is more rapid than cytoplasmic dyes; can be used to test whether compounds are efflux substrates by measuring competition with Nile Red	Does not work well in nonfermenting bacteria such as Pseudomonas spp.; assay optimized for study of AcrAB-TolC	12
1,2′-Dinaphthylamine	Periplasmic; lipophilic dye that fluoresces weakly in aqueous solutions but strongly in nonpolar environments such as the membrane; more lipophilic than Nile Red, so ideal for studying RND efflux pumps because phenylalanines are important for substrate interaction in binding pocket; excitation wavelength, 370 nm; emission wavelength, 810 nm	Most sensitive; can be used to distinguish between efflux rates of AcrB proteins with SNPs;a well retained in membrane while bacteria are in deenergized state; capable of emission in near-infrared region of spectrum, where cellular autofluorescence is low	Does not work well in nonfermenting bacteria such as Pseudomonas spp.	13
Doxorubicin	Fluoresces more extracellularly than intracellularly, so fluorescence increases upon efflux; excitation wavelength, 450 nm; emission wavelength, 600 nm		Very expensive	10, 42

^aSNPs, single-nucleotide polymorphisms.