The role of the methylene blue and toluidine blue monomers and dimers in the photoinactivation of bacteria

The interactions between the phenothiazine dyes, methylene blue (MB) and toluidine blue (TB), and bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Hemophilus influenzae, Escherichia coli and Pseudomonas aeruginosa) were studied spectrophotometrically. This demonstrated that a metachromatic reaction took place between the dyes and bacteria. Furthermore, bacteria induced additional dimerization of MB and TB. The effective dimerization constants of MB and TB were evaluated in the presence of each bacterial strain at a concentration of 108 CFU/ml. The analysis of the effective dimerization constants for MB and TB in the presence of bacteria indicated that the ability to form dimers was greater for TB than for MB. Gram-negative bacteria induced the dye dimerization more intensely than gram-positive bacteria. There was a correlation between the ability of each dye to form dimers in the presence of bacteria and the relative photobactericidal efficacy of each dye against these bacteria. These results provide evidence confirming the essential role of the dye dimers in bacterial photodamage.