Atomic force microscopic study on morphological alterations induced by photodynamic action of Toluidine Blue O in Staphylococcus aureus and Escherichia coli.

Topographical alterations induced by Toluidine Blue O (TBO) mediated photodynamic treatment in Staphylococcus aureus and Escherichia coli was studied using atomic force microscopy (AFM). The AFM images showed distinct differences in the effect of photodynamic treatment on the morphology of S. aureus and E. coli. In S. aureus, photodynamic treatment with TBO resulted in light dose dependent increase in surface bleb formation suggesting breakage in the contact between the cell wall and the membrane with no significant change in the cell dimensions. Photosensitization of E. coli, resulted in surface indentations, significant reduction in the mean cell height, and flattening of bacteria as compared to the bacteria treated with the photosensitizers in the dark. These results indicate damage to the bacterial membrane and reduction of cell volume due to the loss of cytoplasmic materials. Leakage of intracellular contents measured using absorption spectrophotometry was higher and occurred faster in E. coli as compared to S. aureus and correlated with the morphological alterations. The results suggest that with AFM imaging it is possible to distinguish the membranolytic action of TBO in Gram-positive and Gram-negative bacteria.