Fluorometric assessment of Gram-negative bacterial permeabilization

Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted t o an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bact eria. An intact outer membrane is a permeability barrier, and excludes hydr ophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With E scherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphos phate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the t est bacteria. Furthermore, citric acid was shown to be a potent permeabiliz er at millimolar concentrations, its effect being partly (Ps. aeruginosa, S alm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, sugg esting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the N PN uptake assay with the automated spectrofluorometer serves as a convenien t method in analysing and quantifying the effects of external agents, inclu ding potential food preservatives, on Gram-negative bacteria.