Effect of Ca²⁺ on the photobactericidal efficacy of methylene blue and toluidine blue against gram-negative bacteria and the dye affinity for lipopolysaccharides

Background and Objectives: Methylene blue (MB) and toluidine blue (TB) form metachromatic complexes with lipopolysaccharides (LPS). The greater photobactericidal efficacy of TB may be explained by its affinity for LPS. This study aims to elucidate the difference in photobactericidal efficacies between the dyes using Ca²⁺ as a competitor for dye-binding sites on the bacterial outer membrane. Study Design/Materials and Methods: Fixed dye concentration solutions with gram-negative bacteria and increasing concentrations of CaCl₂ were exposed to red laser light. Bacterial survival and spectrophotometry were used to describe the effect of Ca ²⁺ on dye interaction with bacteria and LPS. Results: MB-mediated photokilling was inhibited more significantly than that of TB. CaCl₂ inhibited dye photo-bleaching and suppressed the metachromatic reaction between the dyes and LPS, in particular TB. Conclusions: CaCl₂ inhibits bacterial photokilling by binding with LPS, as well as other anionic polymers including outer membrane proteins. LPS is chiefly involved in TB-mediated photokilling, whereas outer membrane proteins probably are more involved in MB-mediated photokilling.